CN101448858B - Combinatorial therapy involving alpha5beta1 antagonists - Google Patents

Abstract

The present invention relates to the use of VEGF antagonists and alpha5beta1 antagonists for treating cancer and inhibiting angiogenesis and/or vascular permeability, including inhibiting abnormal angiogenesis in diseases. The present invention also relates to use of a VEGFR agonists and alpha5beta1 agonists to promote angiogenesis and vascular permeability. The present invention also relates to new anti-alpha5beta1 antibodies, compositions and kits comprising them and methods of making and using them.

Description

The conjoint therapy that involves alpha 5 beta 1 antagonists

Invention field

The present invention relates to VEGF antagonist and alpha 5 beta 1 antagonists is used for the treatment of cancer and suppresses the blood vessel generation and/or the purposes of vascular permeability (comprising that the abnormal vascular suppressed in disease occurs).The invention still further relates to VEGFR agonist α 5 β 1 agonists for promoting the purposes of blood vessel generation and vascular permeability.The invention still further relates to new anti-alpha 5 beta 1 antibody, the composition that comprises them and test kit, reach preparation and use their method.

Background of invention

The vital role of VEGF-A in pathologic and the generation of non-pathologic vessels obtained establishing fully.Using in vivo VEGF in model induces strong blood vessel to reply that ((1989) EMBO J.8:3801-3808 for Plouet, J etal.; Leung, D.W., etal., (1989) Science 246:1306-1309).Lose single VEGF-A allelotrope and cause embryonic death (Carmeliet, P., etal., (1996) Nature 380:435-439 in mouse; Ferrara, N et al., (1996) Nature 380:439-442).Also know that VEGF is vascular permeability factor, induction of vascular seepage (Senger, D.R.etal., (1995) Science 219:983-985 because it is had the ability; Dvorak, H.F., et al., (1995) Am.J.Pathol.146:1029-1039).So, outside other non-pathologic vessels occurs, VEGF-A involve growth, the occurring with the flesh and blood pipe of reproduction.

VEGF-A can be in conjunction with two kinds of receptor tyrosine kinases (RTK), i.e. VEGFR-1 (Flt-1) and VEGFR-2 (KDR, Flk-1).It is generally acknowledged that VEGFR-2 is the main mediators of mitotic division, blood vessel generation and the permeability reinforcing effect of VEGF-A.In February, 2004, U.S. food and drug administration (FDA) have ratified the chemotherapy regimen that rhuMAb-VEGF (bevacizumab) (the anti-VEGF of a kind of humanization (vascular endothelial growth factor)-A monoclonal antibody) combine based on 5 FU 5 fluorouracil (FU) and have been used for the treatment of metastatic colorectal cancer.Afterwards, FDA had ratified pegaptinib and had been used for the treatment of moist (neovascular) age related macular degeneration (AMD), and pegaptinib a kind ofly can block the fit of 165 amino acid whose VEGF-A isoforms.

Although there are these progressive, many patients with the VEGF antagonist for treating finally die from their disease.Therefore, need the new medicine of exploitation and methods for the treatment of to treat VEGF antagonist therapy is no longer included to the disease that responds or only have partial response.Also need to develop alternative and/or better therapy treat cancer and the disease that worsens, causes or cause because of abnormal vascular.

Summary of the invention

The present invention relates to be used for the treatment of and will benefit from medicine and the method that blood vessel reduces, suffers from the abnormal vascular generation and/or suffers from neoplastic patient.According to an embodiment, the invention provides that blood vessel for suppressing the experimenter occurs and/or the method for vascular permeability, comprise simultaneously or in turn to the VEGF antagonist of experimenter's administering therapeutic significant quantity and the step of alpha 5 beta 1 antagonists.According to another embodiment, the invention provides the method that is used for the treatment of the experimenter who suffers from disease, wherein said experimenter is once to having response by VEGF antagonist for treating disease, but now the VEGF antagonist is partly had response or no longer includes response, described method comprises the step to the alpha 5 beta 1 antagonists of experimenter's administering therapeutic significant quantity.According to another embodiment, the invention provides the method that is used for the treatment of the experimenter who suffers from disease, wherein said disease has resistance or resistance to alpha 5 beta 1 antagonists therapy independent or combined chemotherapy, and described method comprises the VEGF antagonist to experimenter's administering therapeutic significant quantity.

The invention still further relates to new anti-alpha 5 beta 1 antibody, the test kit that comprises them and composition, reach preparation or use their method.According to an embodiment, described new anti-alpha 5 beta 1 antibody is 7H5 antibody described herein or 7H12 antibody or its humanization form or chimeric form.According to another specific embodiment, described 7H5 antibody or 7H12 antibody or its humanization form or chimeric form can be Fab, Fab ', F (ab) ' ₂, scFv (scFv), Fv fragment; The form of double antibody, multi-specificity antibody and linear antibody.According to another embodiment, described new anti-alpha 5 beta 1 antibody can be coupled to another entity, such as, but not limited to therapeutical agent or fluorescence dye or other mark, with detect in the patient or patient's sample in α 5 β 1.This type of new alpha 5 beta 1 antibodies can be used for multiple therapy methods and diagnostic method.For example, this type of anti-alpha 5 beta 1 antibody can be used for treating abnormal vascular generation, tumorigenesis, illness in eye and autoimmune disease.This antibody-like can be used for detecting in the patient or patient's sample in α 5 β 1 protein, its by make in this antibody-like contact patient or patient's sample in α 5 β 1 protein, and qualitatively or quantitatively determine the anti-alpha 5 beta 1 antibody that is bonded to α 5 β 1 protein.

According to another embodiment, the invention provides the method that is used for the treatment of the cancer in the experimenter, comprise simultaneously or use in turn the step of VEGF antagonist and alpha 5 beta 1 antagonists.According to a preferred embodiment, described cancer has response to VEGF antagonist therapy.In another embodiment, the method of the age related macular degeneration (AMD) (comprising moist age related macular degeneration) in the experimenter that treatment suffers from AMD is provided, has comprised simultaneously or the VEGF antagonist of administering therapeutic significant quantity and the step of alpha 5 beta 1 antagonists in turn.In another embodiment, the method for the autoimmune disease in the treatment experimenter is provided, comprise simultaneously or the VEGF antagonist of administering therapeutic significant quantity and the step of alpha 5 beta 1 antagonists in turn.

In one embodiment, at first experimenter to be treated can use the VEGF antagonist, with alpha 5 beta 1 antagonists, treats subsequently.In another embodiment, the experimenter is simultaneously with VEGF antagonist and alpha 5 beta 1 antagonists treatment.According to another embodiment, the experimenter uses the VEGF antagonist for treating, until the experimenter does not respond the VEGF antagonist for treating, then the experimenter treats with alpha 5 beta 1 antagonists.In a specific embodiment, the time standby VEGF antagonist for treating that the experimenter is invasive or early stage in described cancer right and wrong, and standby alpha 5 beta 1 antagonists treatment when described cancer is invasive (invasive).In another embodiment, compare α 5 β 1 levels with rising with the tissue from not suffering from this sick experimenter with the experimenter of alpha 5 beta 1 antagonists treatment in illing tissue.In this case, described method can further comprise the step that detects α 5 β 1 in the experimenter, for example with after the VEGF antagonist for treating in illing tissue.According to an embodiment, described invasive cancer is metastatic carcinoma.According to another embodiment, described early cancer for example is, by the cancer of adjuvant therapy (chemotherapy or excision) treatment.

In a preferred embodiment, described experimenter suffers from and has the disease that abnormal vascular occurs.According to another embodiment, described disease is selected from lower group: cancer, autoimmune disease or illness in eye.According to a preferred embodiment, described disease is selected from lower group: solid tumor, metastatic tumor, soft tissue neoplasm, have a neovascularization disease, there is inflammatory diseases that abnormal vascular occurs, be implanted into the disease occurred after the experimenter and there is the disease of fiber blood vessel (fibrovascular) tissue abnormalities propagation.According to another preferred embodiment, described cancer is selected from lower group: mammary cancer (comprising metastatic breast cancer), cervical cancer, colorectal carcinoma (comprising metastatic colorectal cancer), lung cancer (comprising nonsmall-cell lung cancer), non-hodgkin's (Hodgkin) lymphomas (NHL), chronic lymphocytic leukemia, renal cell carcinoma, prostate cancer (comprising hormone refractoriness prostate cancer (homone refractory prostate cancer)), liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, the soft tissue cancer, gastrointestinal stromal tumor (gastrointestinalstromal tumor), glioblastoma multiforme and multiple myeloma.According to another preferred embodiment, described disease is selected from lower group: macular degeneration (for example moist AMD), diabetic macular edema, rubescent that retinopathy, age bring out, psoriatic, the inflammatory ephrosis, hemolytic uremic syndrome (haemolytic uremic syndrome), diabetic nephropathy (for example proliferative diabetic retinopathy), sacroiliitis (psoriatic arthritis for example, osteoarthritis, rheumatoid arthritis), inflammatory bowel, chronic inflammatory diseases, chronic detachment of retina, chronic uveitis, chronic hyalitis, corneal graft rejection, the cornea neovascularization, the corneal graft neovascularization, Crow grace (Crohn) family name disease, myopia, eye neovascularity disease, Paget (Paget) family name disease, pemphigoid, polyarteritis, laser radiation shape corneal incision is postoperative, the retina neovascularization, Si Yegelun (Sogren) Cotard, ulcerative colitis, transplant rejection, pneumonia, nephrotic syndrome, oedema, the ascites relevant with malignant tumour, apoplexy, hemangiofibroma and neovascular glaucoma.In one embodiment, described experimenter further uses the therapeutical agent that is selected from lower group: antineoplastic agent, chemotherapeutics and cytotoxic agent.

According to a preferred embodiment of the present invention, will suffer from the experimenter of alpha 5 beta 1 antagonists treatment recurrence or become after the VEGF antagonist for treating VEGF antagonist for treating will be tolerated.According to another embodiment, will suffer from metastatic carcinoma with the experimenter of alpha 5 beta 1 antagonists and VEGF antagonist for treating or before treat by adjuvant therapy.In one embodiment, described patient candidate is recurrence, that do not answer for chemotherapeutics (such as irinotecan) or resistance is arranged.The example of this type of disease includes but not limited to metastatic colorectal cancer, recurrent metastatic colorectal cancer, metastatic breast cancer, recurrent metastatic breast cancer, transitivity HER2+ mammary cancer, mammary cancer, HER2+ mammary cancer, transitivity carcinoma of the pancreas, colorectal carcinoma, nonsmall-cell lung cancer, the rectum cancer, nonsmall-cell lung cancer, Metastatic Nsclc, Metastatic carcinoma in the ovary, metastatic renal cell cancer and renal cell carcinoma.

According to an embodiment, the experimenter who suffers from disease described herein is with after VEGF antagonist for treating disease, using supportive care, wherein said supportive care be independent or with the VEGF antagonist in turn or the alpha 5 beta 1 antagonists of simultaneously using.

According to a preferred embodiment, described VEGF antagonist can be selected from lower group: antibody, immunoadhesin, peptide body (peptibody), small molecules and can be under stringent condition and the nucleic acid (for example ribozyme, siRNA and fit) of the making nucleic acid molecular hybridization of coding VEGF.According to a preferred embodiment, described VEGF antagonist is antibody.According to another embodiment, described antibody is monoclonal antibody.According to a preferred embodiment, described VEGF antibody can by

the combination of antibody competition inhibition to people VEGF.According to another embodiment, described VEGF antibody is people's antibody, humanized antibody or chimeric antibody.According to a specific embodiment, described VEGF antibody is

antibody.According to another embodiment, described VEGF antibody is selected from lower group: Fab, Fab ', F (ab) ' ₂, strand FV (scFv), Fv fragment; Double antibody and linear antibody.According to another embodiment, described VEGF antagonist is and to be alpha 5 beta 1 antagonists in conjunction with the bi-specific antibody of VEGF and α 5 β 1.

According to a preferred embodiment, described alpha 5 beta 1 antagonists can be selected from lower group: antibody, immunoadhesin, peptide body, small molecules and can be under stringent condition and the nucleic acid of the making nucleic acid molecular hybridization of coding for alpha 5 β 1.According to a preferred embodiment, described alpha 5 beta 1 antagonists is antibody.According to another embodiment, described antibody is monoclonal antibody.According to another embodiment, described monoclonal antibody is chimeric antibody, such as the anti-human alpha 5 beta 1 antibodies that is called M200 or F200.According to an embodiment, described anti-alpha 5 beta 1 antibody comprises VH sequence SEQ ID NO:1 and VL sequence SEQ ID NO:2.According to another embodiment, described anti-alpha 5 beta 1 antibody comprises sequence SEQ ID NO:3 and sequence SEQ ID NO:4.According to another embodiment, described anti-alpha 5 beta 1 antibody comprises sequence SEQ ID NO:4 and sequence SEQ ID NO:5.According to a preferred embodiment, described anti-alpha 5 beta 1 antibody can the combination to people α 5 β 1 by 7H5 antibody or the inhibition of 7H12 antibody competition.According to a preferred embodiment, described anti-alpha 5 beta 1 antibody is people's antibody, humanized antibody or chimeric antibody.According to a specific embodiment, described anti-alpha 5 beta 1 antibody is 7H5 antibody, 7H12 antibody or its chimeric antibody or humanized antibody.According to another embodiment, described anti-alpha 5 beta 1 antibody is selected from lower group: Fab, Fab ', F (ab) ' ₂, scFv (scFv), Fv fragment; Double antibody and linear antibody.According to another embodiment, described alpha 5 beta 1 antagonists is and to be the VEGF antagonist in conjunction with the bi-specific antibody of VEGF and α 5 β 1.According to another embodiment, described anti-alpha 5 beta 1 antagonist has the effector functions of change.According to an embodiment, described anti-alpha 5 beta 1 antibody changes over cytotoxicity (ADCC) or CDC (CDC) activity (for example, by changing the nucleotide sequence of encoding antibody Fc part) that reduces or stop antibody dependent cellular.According to another embodiment, described anti-alpha 5 beta 1 antibody has changed over and has improved its transformation period (for example, by changing the nucleotide sequence of encoding antibody Fc part) in human body.

According to an embodiment, described VEGF antagonist or alpha 5 beta 1 antagonists are coupled to cytotoxic agent or chemotherapeutics.According to another embodiment, described cytotoxic agent is radio isotope or toxin.

The invention provides the composition that comprises VEGF antagonist, alpha 5 beta 1 antagonists and pharmaceutical acceptable carrier.The present invention also provides the goods that comprise the instruction about detecting α 5 β 1 in the experimenter who has crossed with the VEGF antagonist for treating.

The invention still further relates to the purposes that VEGFR agonist and α 5 β 1 agonists promote blood vessel generation and vascular permeability, and the composition that comprises VEGFR agonist and α 5 β 1 agonists and pharmaceutical acceptable carrier.Described VEGFR agonist and α 5 β 1 agonist conjoint therapies can be used for treatment will benefit from the various diseases that blood vessel occurs and vascular permeability raises, and comprises for example wound healing, such as in treatment chronic wounds, acute wounds and normal wound.

The accompanying drawing summary

Fig. 1 has shown the increase of raising at the stroma cell with express alpha 5 β 1 after VEGF antibody B20-4.1 processing HT29 heterograft tumour.

Fig. 2 has shown that 7H5 and 7H12 antibodies are to the HUVEC cell in direct binding assay.

Fig. 3 shown according to facs analysis, and 7H5 and 7H12 antibodies be to HUVEC, but is not bonded to the RAJI cell.

Fig. 4 has shown that HUVEC adheres to fibronectin when the 7H5 that has purifying and 7H12 monoclonal antibody.

Fig. 5: (A) bar chart has shown 7H5 and the 7H12 impact on HUVEC cell proliferation by total cell count; (B) bar chart has shown 7H5 and the 7H12 impact on HUVEC cell proliferation by the blue dyeing of the Alamar in another assay method.

Fig. 6 is processing with 7H5 latter 0 hour and the photo of HUVEC cell migration in 30 hours, is comparing with negative control (IgG).

The bar chart of Fig. 7 has quantitatively shown the HUVEC cell migration after processing with 7H5 and 7H12

The bar chart of Fig. 8 has shown the per-cent of the HUVEC cell of the Caspase-3 of expressing activation after processing with 7H5 and 7H12 in the apoptosis assay method.

The bar chart of Fig. 9 has shown in the HUVEC caspase 3 with after 7H5 and 7H12 processing/7 activity.

Figure 10 has shown the activity that 7H12 and/or rhuMAb-VEGF are in rabbit ear section wound healing model.

Figure 11 has shown in breast cancer model the result with the mouse of VEGF antibody +/-anti-alpha 5 beta 1 Antybody therapy: (A) figure has shown the class mean gross tumor volume of treated mouse; (B) the Kaplan-Meier graphic representation has shown the per-cent function in time that stays animal under study for action.Animal meets or exceeds 1500mm in its tumour ³the time exit research.

Figure 12 has shown in model of colon cancer the result with the mouse of VEGF antibody +/-anti-alpha 5 beta 1 Antybody therapy: (A) figure has shown the class mean gross tumor volume of treated mouse; (B) the Kaplan-Meier graphic representation has shown the per-cent function in time that stays animal under study for action.Animal meets or exceeds 1500mm in its tumour ³the time exit research.

Figure 13 has shown the result of the mouse for the treatment of with anti-alpha 5 beta 1 antibody or chemotherapeutics in model of colon cancer: (A) figure has shown the class mean gross tumor volume of treated mouse; (B) the Kaplan-Meier graphic representation has shown the per-cent function in time that stays animal under study for action.Animal meets or exceeds 1500mm in its tumour ³the time exit research.

Figure 14 has shown that being bonded to R9ab (a kind of rabbit fibroblast clone) goes up α 5 β's 1 ¹²⁵the scatchard curve of I-7H5.

Figure 15 has shown that being bonded to R9ab (a kind of rabbit fibroblast clone) goes up α 5 β's 1 ¹²⁵the scatchard curve of I-7H12.

Figure 16 has shown the result of the anti-alpha 2 integrin α 5 β 1 IgG epitope mappings of multiple anti-alpha 5 beta 1 antibody/competitive binding assay method.

Detailed Description Of The Invention

Be not bound by theory, we advise that stroma cell raises the increase meeting other angiogenesis factor is brought to disease sites, thus the loss of VEGF activity in the patient of VEGF antagonist therapy for treating for compensation.Can cause the minimizing of stroma cell with the stroma cell of anti-alpha 5 beta 1 antibody target express alpha 5 β 1, thereby reduce the generation of potential compensatory angiogenesis factor.Perhaps/in addition, we advise suppressing endothelium-extracellular matrix and interact, and particularly suppress α 5 β 1 binding interactions and will return and strengthen VEGF antagonist therapy by suppressing extracellular matrix track that blood vessel occurs to stay along the decline blood vessel that cause due to VEGF antagonist therapy.Therefore, with any VEGF antagonist for treating simultaneously or carry out the alpha 5 beta 1 antagonists treatment and can suppress blood vessel from this VEGF antagonist for treating recovery any VEGF antagonist for treating after, thereby the neovascularity growth recovery.

" α 5 β 1 " or " a5 β 1 " refer to the integrin that comprises two different proteins (being subunit α 5 and β 1).α 5 β 1 have demonstrated in conjunction with fibronectin, L1-CAM and Fibrinogen.Alpha 5 beta 1 integrin also is called VLA-5 (very late activation-5), alpha5betal, CD49e/CD29, fibronectin receptor, FNR and GPIc-IIa.According to a preferred embodiment, described α 5 β 1 are people α 5 β 1.

" α 5 ", also referred to as IC subunit and the FNR α chain of CD49e, alpha5, integrin Alpha 5 subunit, VLA-5 α subunit, GPIc-IIa, it has four kinds of isoforms that generate by alternative splicing (isoform) (A-D).They are different in its cytoplasmic structure territory.The aminoacid sequence of α 5 people's isoforms can see respectively for example Genbank numbering: X07979, U33879, U33882 and U33880.

" β 1 " is also referred to as CD29, betal, thrombocyte GPIIa; VLA-β chain; β-1 integrin chain, CD29; FNRB; MDF2; VLAB; GPIIA; MSK12 and VLA5B.The aminoacid sequence of people β 1 is found in for example Genbank numbering X06256.

Term " VEGF " or " VEGF-A " refer to 165 amino acid whose human vascular endothelial growth factors and 121,189 relevant and 206 amino acid whose human vascular endothelial growth factors for this paper the time, as Mol.Endocrin such as the Science 246:1306 (1989) such as Leung and Houck, 5:1806 (1991) is described, and natural allelic form and the form processing of existing.Term " VEGF " also refers to the VEGF from inhuman species such as mouse, rat or primate.Sometimes, from the VEGF of specific species, be expressed as follows, hVEGF means people VEGF, and mVEGF means mouse VEGF, etc.Term " VEGF " also is used in reference to the amino acid 8-109 position that comprises 165 amino acid whose human vascular endothelial growth factors or the clipped form polypeptide of 1-109 position.May be by for example " VEGF (8-109) ", " VEGF (1-109) " or " VEGF in the application ₁₆₅" differentiate any this type of form VEGF.The amino acid position of " brachymemma " natural VE GF is numbered as shown in natural VE GF sequence.For example, the 17th amino acids (methionine(Met)) in the natural VE GF of brachymemma is also the 17th (methionine(Met)) in natural VE GF.The natural VE GF of brachymemma has the binding affinity to KDR and Flt-1 acceptor suitable with natural VE GF.According to a preferred embodiment, described VEGF is people VEGF.

" VEGF antagonist " refers to can to neutralize, block, suppress, eliminate, reduce or disturb the VEGF molecule of active (comprise its be combined with VEGF or one or more vegf receptors or their nucleic acid of encoding).Preferably, described VEGF antagonist can be in conjunction with VEGF or vegf receptor.The VEGF antagonist comprise VEGF antibody and Fab thereof, can in conjunction with the micromolecular inhibitor of the polypeptide (for example immunoadhesin, peptide body) of VEGF and vegf receptor block ligand-acceptor interaction, anti-vegf receptor antibody and vegf receptor antagonist such as VEGFR Tyrosylprotein kinase, can for example, in conjunction with the fit of VEGF and can be under stringent condition and the nucleic acid (RNAi) of coding VEGF or vegf receptor hybridization.According to a preferred embodiment, described VEGF antagonist can and suppress the endothelial cell proliferation (in vitro) that VEGF induces in conjunction with VEGF.According to a preferred embodiment, described VEGF antagonist with the avidity that is greater than non-VEGF or non-vegf receptor in conjunction with VEGF or vegf receptor.According to a preferred embodiment, described VEGF antagonist with the Kd between 1uM and 1pM in conjunction with VEGF or vegf receptor.According to another preferred embodiment, described VEGF antagonist with the Kd between 500nM and 1pM in conjunction with VEGF or vegf receptor.

According to a preferred embodiment, described VEGF antagonist is selected from lower group: polypeptide such as antibody, peptide body, immunoadhesin, small molecules or fit.In a preferred embodiment, described antibody be VEGF antibody (such as

antibody) or anti-vegf receptor antibody (such as anti-VEGFR2 or anti-VEGFR3 antibody).Other example of VEGF antagonist comprises: VEGF-Trap, Mucagen, PTK787, SU11248, AG-013736, Bay 439006 (sorafenib), ZD-6474, CP632, CP-547632, AZD-2171, CDP-171, SU-14813, CHIR-258, AEE-788, SB786034, BAY579352, CDP-791, EG-3306, GW-786034, RWJ-417975/CT6758 and KRN-633.

" VEGF antibody " refers to the antibody with enough avidity and specific binding VEGF.Preferably, VEGF antibody of the present invention can be at target and is intervened in the disease wherein involve the VEGF activity or illness and be used as therapeutical agent.VEGF antibody usually can be in conjunction with other VEGF homologue, such as VEGF-B or VEGF-C, and can be in conjunction with other somatomedin, such as PlGF, PDGF or bFGF yet.A kind of preferred VEGF antibody is the monoclonal anti VEGF antibody A 4.6.1 that generates with hybridoma ATCC HB 10709 monoclonal antibody in conjunction with identical epi-position.More preferably, VEGF antibody is the anti-VEGF monoclonal antibody of recombinant humanized generated according to (1997) Cancer Res.57:4593-4599 such as Presta, includes but not limited to be called rhuMAb-VEGF (bevacizumab; BV;

) antibody.According to another embodiment, operable VEGF antibody includes but not limited to the antibody disclosed in WO 2005/012359.According to an embodiment, variable region of heavy chain and the variable region of light chain of the arbitrary antibody (for example G6, G6-23, G6-31, G6-23.1, G6-23.2, B20, B20-4 and B20.4.1) disclosed in Figure 24 that described VEGF antibody comprises WO 2005/012359,25,26,27 and 29.In another preferred embodiment, the VEGF antibody that is called ranibizumab is such as diabetic neuropathy and AMD and the VEGF antagonist of using for illness in eye.

VEGF antibody " rhuMAb-VEGF " (bevacizumab, BV), also referred to as " rhuMAb VEGF " or " ", be the anti-VEGF monoclonal antibody of recombinant humanized generated according to Presta et al. (1997) Cancer Res.57:4593-4599.Human IgG1's framework region that it comprises sudden change and come self energy blocking-up people VEGF in conjunction with the antigen of the mouse-anti hVEGF monoclonal antibody A4.6.1 of its acceptor in conjunction with complementary determining region.The aminoacid sequence of rhuMAb-VEGF about 93% (comprising most of framework region) derived from human IgG1, and about 7% sequence is derived from murine antibody A4.6.1.RhuMAb-VEGF has approximately 149,000 daltonian molecular weight, and is glycosylated.Other VEGF antibody comprises U.S. Patent No. 6,884,879 and WO2005/044853 in the antibody put down in writing.

VEGF antibody Ranibizumab or antibody or rhuFab V2 are anti-human VEGF Fab fragments humanized, affinity maturation.Ranibizumab generates in coli expression carrier and fermentation using bacteria by the standard technological method of recombinating.Ranibizumab is not glycosylated and molecular weight is about 48,000 dalton.Referring to WO98/45331 and US20030190317.

" alpha 5 beta 1 antagonists " refers to suppress any molecule of the biologic activity of α 5 β 1.According to a preferred embodiment, described antagonist molecules energy specific binding α 5 β 1.According to a preferred embodiment, described antagonist molecules can be in conjunction with α 5.According to a preferred embodiment, alpha 5 beta 1 antagonists with the avidity larger with respect to non-alpha 5 beta 1 integrin preferentially in conjunction with α 5 β 1.According to a preferred embodiment, described antagonist is selected from lower group: can suppress polypeptide such as antibody, peptide body or immunoadhesin, the small molecules or fit of α 5 β 1 in conjunction with its part (particularly fibronectin), or can be under stringent condition and the nucleic acid (RNAi that for example can disturb α 5 to express) of the making nucleic acid molecular hybridization of coding for alpha 5 β 1.The biologic activity of α 5 β 1 can be to be selected from arbitrary effect, effect combination or all effects of lower group: (1) is in conjunction with fibronectin; (2) strengthen the migration of cell on fibronectin; (3) improve the survival of cell when having fibronectin that comprises α 5 β 1; (4) improve the propagation of cell when having fibronectin that comprises α 5 β 1; (5) improving the pipe of cell when having fibronectin that comprises α 5 β 1 forms.

The example of anti-alpha 5 beta 1 antagonistic antibodies comprises M200 and F200 (WO 2004/089988A2), 7H5 antibody described herein and 7H12 antibody and chimeric antibody, fully human antibodies and humanized antibody.For example, M200 and F200 antibody can be derived from variable heavy chain and the variable light chains of mouse anti human alpha 5 beta 1 antibodies IIA1 (Pharmingen, San Diego, Ca).The example of α 5 β 1 micromolecular inhibitors comprises Ac-PHSCN-NH2 (WO-9822617A1) and (S)-2-[(2; 4; the 6-trimethylphenyl) alkylsulfonyl] amino-3-[7-carbobenzoxy-(Cbz)-8-(2-pyridinylamino methyl)-1-oxa--2; the 7-diamino is for spiral shell-(4,4)-one-2-alkene-3-yl] carbonylamino] propionic acid.According to a preferred embodiment, described alpha 5 beta 1 antagonists is in conjunction with α 5 β 1 and not in conjunction with α V β 3 or α V β 5 or α V β 1.According to a preferred embodiment, described alpha 5 beta 1 antagonists with the Kd between 1uM and 1pM in conjunction with α 5 β 1.According to another preferred embodiment, described alpha 5 beta 1 antagonists with the Kd between 500nM and 1pM in conjunction with α 5.According to a preferred embodiment, described alpha 5 beta 1 antibodies is can be in the competitive binding assay method and the antibody of 7H5 antibody or 7H12 antibody competition α 5 β 1 combinations.According to another preferred embodiment, described antibody is can be by the antibody of antibody competition inhibition α 5 β 1 combinations that generated by following hybridoma: on March 7th, 2006 is as the hybridoma of A5/ β 1 7H5.4.2.8 (ATCC No.PTA-7421) preservation or as the hybridoma of A5/ β 1 7H12.5.1.4 (ATCC No.PTA-7420) preservation.

The molecule that " VEGFR agonist " refers to activate vegf receptor or improve its expression.The VEGFR agonist includes but not limited to for example ligand agonist, VEGF variant, antibody and the active fragments of VEGFR.

" α 5 β 1 agonists " refer to activate α 5 β 1 or improve the molecule of its expression.α 5 β 1 agonists include but not limited to for example ligand agonist of α 5 β 1.

Take and be bonded to molecule (such as antibody) overlapping on the target thing or that similar zone is feature and can identify by competitive inhibition/binding assay.

In one embodiment, use HUVEC or other cell of express alpha 5 β 1 in the competitive inhibition assay method, and assess two kinds of anti-alpha 5 beta 1 antibody binding site relative to each other with FACS.For example, the HUVEC cell can be cleaned in tapered tube, and with 1000rpm centrifugal 5 minutes.Usually by twice of sediment undergoes washing.Then, can cell is resuspended, counting, and be placed on ice until use.Can in hole, add 100 μ l the first anti-alpha 5 beta 1 antibody (for example, since 1 μ g/ml concentration or lower concentration).Then, can for example, to adding 100 μ l cells (20x10 in each hole ⁵individual cell), and incubation on ice 30 minutes.Then, can be to adding 100 μ l biotinylation anti-alpha 5 beta 1 antibody (5 μ g/ml liquid storage) in each hole, and incubation on ice 30 minutes.Then cell is cleaned, and with 1000rpm centrifugal 5 minutes.Suck supernatant liquor.Xiang Kongzhong adds second antibody R-PE coupling streptavidin (Jackson 016-110-084) (100 μ l, 1:1000).Then, plate can be wrapped in tinsel, and incubation on ice 30 minutes.After incubation, can be by sediment undergoes washing, and with 1000rpm centrifugal 5 minutes.Can throw out is resuspended, and be transferred to microburette, for facs analysis.

" angiogenesis factor " or " blood vessel propellant " refers to stimulate vascular development, such as promoting blood vessel that the somatomedin of (angiogenesis), endothelial cell growth, stabilization of vascular and/or vasculogenesis (vasculogenesis) etc. occurs.For example, angiogenesis factor includes but not limited to for example member of VEGF and VEGF family, PlGF, PDGF family, fibroblast growth family (FGF), TIE part (angiogenin), ephrin, Del-1, acid (aFGF) and alkalescence (bFGF) fibroblast growth factor, follistatin (Follistatin), granulocyte colony-stimulating factor (G-CSF), pHGF (HGF)/scattering factor (SF), interleukin-8 (IL-8), Leptin, Midkine, placenta growth factor, thymidine phosphorylase/platelet-derived endothelial cell growth factor (PD-ECGF), Thr6 PDGF BB is PDGF-BB or PDGFR-β especially, Pleiotrophin (PTN), Progranulin, Proliferin, transforminggrowthfactor-α (TGF-α), transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) etc.It also comprises the factor of accelerating wound healing, such as the member of tethelin, insulin like growth factor-1 (IGF-I), VIGF, Urogastron (EGF), CTGF and family thereof, and TGF-α and TGF-β.Referring to for example Klagsbrun and D ' Amore, Annu.Rev.Physiol.53:217-39 (1991); Streit and Detmar, Oncogene 22:3172-3179 (2003); Ferrara & Alitalo, Nature Medicine 5 (12): 1359-1364 (1999); Tonini etc., Oncogene 22:6549-6556 (2003) (for example enumerating the table 1 of known angiogenesis factor); Sato, Int.J.Clin.Oncol.8:200-206 (2003).

In a preferred embodiment, according to " Kd " or " Kd value " of VEGF antibody of the present invention, be that the radio-labeling VEGF binding assay (RIA) that antibody and VEGF molecule by the described use of following assay method Fab pattern carry out is measured: under the condition by the titration series there being unmarked VEGF, with Cmin ( ¹²⁵i) mark VEGF (109) balance Fab, then with anti-Fab antibody, the VEGF of coated flat board seizure institute combination measures the solution binding affinity (Chen, et al., (1999) J Mol Biol 293:865-881) of Fab to VEGF.In order to determine condition determination, catch with the coated microtiter plate (Dynex) of anti-Fab antibody (Cappel Labs) and spend the night with 5 μ g/ml in 50mM sodium carbonate (pH 9.6), use subsequently 2% in PBS (w/v) bovine serum albumin(BSA) in room temperature (approximately 23 ℃) sealing 2-5 hour.In non-absorption dull and stereotyped (Nunc#269620), by 100pM or 26pM[ ¹²⁵i]-Fab interested of VEGF (109) and serial dilution, for example Fab-12 (Presta et al., (1997) Cancer Res.57:4593-4599) mixes.Then by Fab incubated overnight interested; But, be incubated sustainable 65 hours to guarantee to reach balance.After this, mixture is transferred to and catches plate to carry out room temperature insulation 1 hour.Then remove solution, and wash plate 8 times with the PBS containing 0.1%Tween-20.After dull and stereotyped drying, add 150 μ l/ hole scintillation solution (MicroScint-20; Packard), then upper to plate count 10 minutes in Topcount gamma counter (Packard).Select each Fab to provide to be less than or equal to 20% concentration of maximum combined for the competitive binding assay method.According to another embodiment, Kd or Kd value are to use BIAcore by surperficial plasmon resonance assay method ^tM-2000 or BIAcore ^tM-3000 (BIAcore, Inc., Piscataway, NJ) are used immobilization hVEGF (8-109) CM5 chips to measure in about 10 response units (RU) in 25 ℃.In brief, hydrochloric acid N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimide (EDC) for specification sheets and N-hydroxy-succinamide (NHS) activation carboxymethylation dextran biologic sensor chip (CM5, BIAcore Inc.) according to supplier.With 10mM sodium acetate pH 4.8, people VEGF is diluted to 5 μ g/ml (approximately 0.2 μ M), then with the flow velocity of 5 μ l/ minutes, is injected into and obtains the approximately coupling protein matter of 10 response units (RU).After injecting people VEGF, inject the 1M thanomin with sealing unreacted group.In order to carry out kinetic measurement, in 25 ℃ of flow velocitys with about 25ul/ minute, be infused in the Fab (0.78nM to 500nM) of twice serial dilution in the PBS (PBST) containing 0.05%Tween-20.Use simply one to one Lang Gemiaoer (Langmuir) combination model (BIAcore Evaluation Software version 3.2) by while matching combination and the sensing figure calculations incorporated speed (k that dissociates _on) and the speed (k that dissociates _off).Equilibrium dissociation constant (Kd) is with ratio k _off/ k _oncalculate.Referring to for example Chen, Y., et al., J Mol Biol 293:865-881 (1999).If, according to surperficial plasmon resonance assay method above, association rate surpasses 10 ⁶m ^-1s ^-1association rate can be measured by the fluorescent quenching technology so, according to using the measurement of stirring cuvette in spectrometer such as the spectrophotometer that has been equipped with cut-off device (astop-flow equipped spectrophometer) (Aviv Instruments) or 8000 serial SLM-Aminco spectrophotometers (ThermoSpectronic), under the condition that has the cumulative short type of people VEGF (8-109) of concentration or mouse VEGF, measure PBS, the 20nM VEGF antibody in pH 7.2 (Fab form) (excites=295nm in the fluorescent emission intensity of 25 ℃; Emission=340nm, 16nm band is logical) rising or reduction.Can implement as the target thing Kd that similar binding assay is measured anti-alpha 5 beta 1 Fab or antibody with α 5 β 1.

For this paper the time, experimenter to be treated refers to Mammals (such as people, non-human primates, rat, mouse, ox, horse, pig, sheep, goat, dog, cat etc.).The experimenter can be clinical patients, clinical trial volunteer, laboratory animal etc.The experimenter suspects to suffer from or risky any Other diseases of suffering from cancer, immunological disease or having the abnormal vascular generation, diagnoses and cancer, immunological disease is arranged or have any Other diseases that abnormal vascular occurs.Cancer, immunological disease or show many diagnostic methods of any Other diseases that abnormal vascular occurs and the clinical description of those diseases is known in the art.According to a preferred embodiment, remain to refer to the people according to the experimenter of the present invention's treatment.

For example, when the term abnormal vascular betides neovascularity overgrowth in morbid state or that cause morbid state or improper (seeing bad blood vessel occurrence positions, time or startup from the medical science viewpoint).Excessive, improperly or blood vessel out of control betide and facilitate morbid state to worsen or while causing the neovascularity growth of morbid state, (comprise colorectal carcinoma such as cancer especially vascularization solid tumor and metastatic tumor, lung cancer (especially small cell lung cancer), or prostate cancer), the disease caused by the eye neovascularization is diabetic blindness especially, retinopathy, the macular degeneration (AMD) that primary diabetes mellitus retinopathy (primarily diabeticretinopathy) or age bring out, choroid neovascularization (CNV), diabetic macular edema, pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), the cornea neovascularization, retina neovascularization and rubescent, psoriatic, psoriatic arthritis, hemangioblastoma are such as vascular tumor, the inflammatory ephrosis, such as glomerulonephritis, especially membrano proliferative glomerulonephritis, hemolytic uremic syndrome (haemolytic uremicsyndrome), diabetic nephropathy or hypertensive nephrosclerosis, various inflammatory diseasess, such as sacroiliitis, disease, endometriosis or chronic asthma that especially rheumatoid arthritis, inflammatory bowel, psoriatic, sarcoidosis, arteriosclerosis and transplanting occur afterwards, and surpass 70 kinds of other illness.Neovascularity can be supplied illing tissue, destroy healthy tissues, and, in the situation of cancer, neovascularity tolerable tumour cell is escaped circulation and rested on (metastases) in other organ.The patient of the above-mentioned disease of those risky generations for the treatment of is contained in the present invention.

As other patients of the candidate of accepting antibody of the present invention or polypeptide, have or risky generation fiber vascular tissue abnormality proliferation, rosacea (acne erythematosa), acquired immune deficiency syndrome (AIDS), obstruction of artery, atopy keratitis, bacterial canker, Behcets disease (Bechets disease), the tumour (blood borne tumor) that blood is propagated, the carotid artery obstruction disease, the choroid neovascularization, chronic inflammatory diseases, chronic detachment of retina, chronic uveitis, chronic hyalitis, contact lense is worn excessively, corneal graft rejection, the cornea neovascularization, the corneal graft neovascularization, Crohn disease (Crohn ' s disease), eales disease (Eales disease), epidemic keratoconjunctivitis, mycotic ulcer, herpes simplex infection, zoster infects, hyperviscosity syndrome, Kaposi sarcoma (Kaposi ' ssarcoma), leukemia, the lipid sex change, Lai Mushi sick (Lyme ' s disease), edge keratolysis (marginal keratolysis), mooren's ulcer (Mooren ulcer), mycobacterial infections except leprosy, myopia, ocular neovascular disorders, depending on nest (optic pits), Ao-Wei bis-Cotards (Osler-Weber syndrome) (Ao Sile-weber-Lang Di (Osler-Weber-Rendu) disease), osteoarthritis, osteitis deformans (Pagetsdisease), pars planitis (pars planitis), pemphigoid, phylectenulosis, polyarteritis, laser infectious-related complication (post-laser complication), protozoal infections, pseudoxanthoma elasticum, pteryium (pterygium), keratitis sicca, radial keratotomy, the retina neovascularization, retinopathy of prematurity, Terry's sign, sarcoid/sarcoidosis (sarcoid), scleritis, sicklemia, siogren's syndrome (Sogrens syndrome), solid tumor, recessive macular dystrophy (Stargarts disease), Yue-Si sick (Steven ' s Johnson disease), upper limb keratitis (superior limbic keratitis), syphilis, the general lupus, special Li Angshi rim degeneration (Terrien ' smarginal degeneraion), toxoplasmosis, wound, the tumour of Ewing sarcoma (Ewing sarcoma), the tumour of neuroblastoma, osteosarcomatous tumour, the tumour of retinoblastoma, the tumour of rhabdosarcoma, ulcerative colitis, vein obstruction, vitamin A deficiency and Wegener sarcoidosis (Wegenerssarcoidosis), the bad blood vessel relevant with diabetes occurs, parasitosis, abnormal wound healing, wound, loose after damage or operation, hair growth suppresses, ovulation and corpus luteum form and suppress, in uterus, fetal development suppresses and implants and suppress.

Anti-angiogenic generation therapy can be used for the general treatment of the following: transplant rejection, pneumonia, nephrotic syndrome, preeclampsia, pericardial effusion (such as relevant with pericarditis) and hydrothorax, the disease that is characterized as bad vascular permeability and illness, the for example oedema relevant with brain tumor, the ascites relevant with malignant tumour, plum Ge Sishi (Meigs) syndrome, pneumonia, nephrotic syndrome, pericardial effusion, hydrothorax, the permeability relevant with cardiovascular disorder, such as the illness after myocardial infarction and apoplexy, like that.

Comprise hemangiofibroma (tending to hemorrhage abnormal vascular) according to other blood vessel generation dependence disease of the present invention, neovascular glaucoma (angiogenic growth in eye), arteriovenous malformotion (the abnormal contact between artery and vein), unconnected fracture (fracture that can not heal), atherosclerotic plaque (sclerosis of artery), botryomycosis hominis (the common skin injury formed by blood vessel), scleroderma (a kind of form of connective tissue disease), vascular tumor (tumour formed by blood vessel), trachoma (first reason that the third world is blind), bleeder's joint, blood vessel adhesion and hypertrophic cicatrix (abnormal scar formation).

" treatment " refer to therapeutic treatment and preventative or precaution measure the two.Need the experimenter for the treatment of to comprise the experimenter who suffers from for a long time illness and the experimenter that will prevent illness.

Term " recurrence " refers to that cancer or disease return after the clinical assessment of disappearance of disease.Long distance shifts or the diagnosis of local recurrence can be considered as recurrence.

Term " tolerance ", " resistance ", " resistance " or " refractoriness " refer to that cancer or disease are to not response for the treatment of.

Term " adjuvant therapy " refers to the treatment given afterwards in main therapy (normally operation).The adjuvant therapy of cancer or disease can comprise immunotherapy, chemotherapy, radiotherapy or hormonotherapy.

Term " supportive care " refers to give in order to help to maintain previous result for the treatment of plannedly controls again.Usually in order to help, cancer to be remained on and disappears or extend response to specific therapy (no matter progression of disease) and give supportive care.

Term " invasive cancer " refers to spread into over its initial organized layer the cancer of normal surrounding tissue.The invasive cancer is can yes or no metastatic.

Term " Noninvasive cancer " refers to very early stage cancer or does not propagate the cancer that surpasses tissue of origin.

Term " progresson free survival " during oncology middle finger treatment and afterwards cancer not have the time span of growing.Progresson free survival comprises that patient experience responds fully or the time quantum of partial response, and patient experience is stablized the time quantum of the state of an illness.

Term " PD " can refer to start to surpass 20% tumor growth-or due to the increase of quality or due to the diffusion of tumour from treatment in oncology.

" illness " refers to that any meeting benefits from the illness of Antybody therapy.For example, suffer from maybe need to prevent abnormal vascular occur (excessive, improperly or blood vessel out of control occur) or the Mammals of vascular permeability.This comprises chronic and acute disease or disease, comprises that those make Mammals tend to the pathological condition of discussed illness.The non-limitative example of illness to be treated comprises pernicious and innocent tumour herein; Non-leukemia and lymph sample malignant tumour; Neurone, neuroglia, astrocyte, hypothalamus and other body of gland, scavenger cell, epithelium, matrix and segmentation cavity illness; And inflammatory, blood vessel generation and immunology illness.

Term " cancer " and " carcinous " refer to or describe feature in Mammals be typically the not modulated physiology illness of Growth of Cells.The example of cancer includes but not limited to cancer (cancer knurl), lymphoma, blastoma, sarcoma and leukemia.The more specifically example of this type of cancer comprises squamous cell carcinoma, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer, hepatic carcinoma), bladder cancer, hepatoma (hepatoma), mammary cancer, colorectal carcinoma, colorectal carcinoma, carcinoma of endometrium, salivary-gland carcinoma, kidney (kidney cancer, renal cancer), prostate cancer, carcinoma vulvae, thyroid carcinoma, head and neck cancer, the rectum cancer, colorectal carcinoma, lung cancer (comprises small cell lung cancer, nonsmall-cell lung cancer, the squama cancer of the gland cancer of lung and lung), squamous cell carcinoma (for example epithelium squamous cell carcinoma), prostate cancer, peritoneal cancer, hepatocellular carcinoma, cancer of the stomach (gastric or stomach cancer) (comprising gastrointestinal cancer), carcinoma of the pancreas, glioblastoma, retinoblastoma, astrocytoma, thecoma, gynandroblastoma, hepatoma (hepatoma), haematological malignancies (comprises non-hodgkin's (Hodgkin) lymphomas (NHL), multiple myeloma and acute haematological malignancies), carcinoma of endometrium or uterus carcinoma, endometriosis, fibrosarcoma, choriocarcinoma, salivary-gland carcinoma, carcinoma vulvae, thyroid carcinoma, the esophageal carcinoma, liver cancer (hepatic carcinoma), anus cancer, penile cancer, nasopharyngeal carcinoma, laryngocarcinoma, Ka Boxi (Kaposi) sarcoma, melanoma, skin carcinoma, Schwann-cell tumor, oligodendroglioma, neuroblastoma, rhabdosarcoma, osteogenic sarcoma, leiomyosarcoma, urethral carcinoma, thyroid carcinoma, Willms (Wilm) family name tumour, and B cell lymphoma (comprises rudimentary/folliculus non_hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, middle rank/folliculus NHL, middle rank diffusivity NHL, senior immunoblast NHL, senior lymphocytoblast property NHL, senior small non-cleaved cell NHL, thesaurismosis (bulky disease) NHL, lymphoma mantle cell, the AIDS associated lymphoma, with Walden Si Telunshi (Waldenstrom) macroglobulinemia), lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell, chronic myeloblastosis, with lymphocytic hyperplasia venereal disease disease (PTLD) after transplanting, and plum Ge Sishi (Meigs) syndrome, the abnormal vascular propagation relevant with phakomatoses (phakomatoses).

" tumour " refers to the growth of all neoplastic cells and propagation for this paper the time, no matter is pernicious or optimum, and before all cancers and cell and tissue cancer.

Term " anti-tumor compositions " or " antineoplastic agent " refer to can be used for treating the composition of cancer, and it comprises at least one active therapeutic agent, for example " carcinostatic agent ".The medicament that the example of therapeutical agent (carcinostatic agent) includes but not limited to use in for example chemotherapeutics, growth inhibitor, cytotoxic agent, radiotherapy, antiangiogenic agent, apoptosis agent, antitublin and other are used for the treatment of medicament such as anti-HER-2 antibody, anti-CD20 antibodies, EGF-R ELISA (EGFR) antagonist (for example tyrosine kinase inhibitor), the HER1/EGFR inhibitor (erlotinib (Tarceva for example of cancer ^tM), Thr6 PDGF BB inhibitor (Gleevec for example ^tM(Imatinib Mesylate)), cox 2 inhibitor (such as celecoxib (celecoxib)), Interferon, rabbit, cytokine, the antagonist (such as neutrality antibody) of can one or more target things in ErbB2, ErbB3, ErbB4, PDGFR-β, BAFF, BR3, APRIL, BCMA or vegf receptor being combined, TRAIL/Apo2, and other biological activity and organic chemistry agent etc.Their combination is also contained in the present invention.

" growth inhibitor " refers in vitro and/or cytostatic compound or composition in vivo for this paper the time.So, growth inhibitor can be significantly to reduce the medicament of the cell per-cent in the S phase.The example of growth inhibitor comprises the cell cycle the advance medicament of (position beyond the S phase) of blocking-up, such as inducing the medicament that G1 stagnates and the M phase stagnates.Classical M phase blocker comprise Changchun medicine class (vincas) (vincristine(VCR) (vincristine) and vinealeucoblastine(VLB) (vinblastine)), , and Topoisomerase II inhibitors such as Dx (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), Etoposide (etoposide) and bleomycin (bleomycin).Medicaments of those retardances G1 also overflow and enter the S phase and stagnate, for example DNA alkylating agent class such as tamoxifen (tamoxifen), prednisone (prednisone), Dacarbazine (dacarbazine), chlormethine (mechlorethamine), cis-platinum (cisplatin), Rheumatrex (methotrexate), 5 FU 5 fluorouracil (5-fluorouracil) and ara-C.More information can be referring to The Molecular Basis of Cancer, Mendelsohn and Israel compiles, the 1st chapter, be entitled as " Cell cycle regulation, oncogenes, and antineoplastic drugs ", Murakamiet al., WB Saunders, Philadelphia (1995), especially the 13rd page.

Term " cytotoxic agent " refers to suppress or stops cell function and/or cause the material of cytoclasis for this paper the time.This term intention comprises radio isotope (I for example ¹³¹, I ¹²⁵, Y ⁹⁰and Re ¹⁸⁶), the enzyme of chemotherapeutics and toxin such as bacterium, fungi, plant or animal origin live toxin or its fragment.

" chemotherapeutics " refers to can be used for treating the chemical compound of cancer.The example of chemotherapeutics comprises the chemical compound that can be used for treating cancer.The example of chemotherapeutics comprises alkylating agent class (alkylating agents), such as phosphinothioylidynetrisaziridine (thiotepa) and

endoxan (cyclophosphamide), alkyl sulfonate esters class (alkyl sulfonates), such as busulfan (busulfan), Improsulfan (improsulfan) and piposulfan (piposulfan), aziridines (aziridines), such as Benzodepa (benzodepa), card ripple quinone (carboquone), U.S. appropriate in sending (meturedepa) and uredepa (uredepa), Ethylenimine class (ethylenimines) and methylmelamine class (methylamelamines), comprise hemel (altretamine), triethylenemelamine (triethylenemelamine), APO (triethylenephosphoramide), TESPA (triethylenethiophosphoramide) and trimethylolmelamine (trimethylolomelamine), Annonaceousacetogenicompounds (acetogenins) (especially bullatacin (bullatacin) and bullatacin ketone (bullatacinone)), camptothecine (camptothecin) (comprising synthetic analogues Hycamtin (topotecan)), bryostatin (bryostatin), callystatin, CC-1065 (comprising its Adozelesin (adozelesin), Carzelesin (carzelesin) and Bizelesin (bizelesin) synthetic analogues), hidden algae element class (cryptophycins) (particularly hidden algae element 1 and hidden algae element 8), dolastatin (dolastatin), duocarmycin (comprising synthetic analogues, KW-2189 and CB1-TM1), Eleutherobin (eleutherobin), pancratistatin, sarcodictyin, sponge chalone (spongistatin), nitrogen mustards (nitrogen mustards), such as Chlorambucil (chlorambucil), Chlornaphazine (chlornaphazine), courage phosphamide (cholophosphamide), Estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine), mustron (mechlorethamine oxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), Trofosfamide (trofosfamide), uracil mastard (uracil mustard), nitrosourea (nitrosoureas), such as BCNU (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine) and Ranimustine (ranimustine), antibiotics, for example, such as Enediyne Antibiotic (enediyne) (Calicheamicin (calicheamicin), especially Calicheamicin γ lI and Calicheamicin ω I1 (referring to for example Agnew, Chem.Intl.Ed.Engl.33:183-186 (1994)), anthracycline antibiotic (dynemicin), comprise dynemicinA, diphosphonates (bisphosphonates), such as clodronate (clodronate), Ai Sibo mycin (esperamicin), and Neocarzinostatin (neocarzinostatin) chromophore and related colour albumen Enediyne Antibiotic chromophore), aclacinomycin (aclacinomycin), D actinomycin D (actinomycin), anthramycin (anthramycin), azaserine (azaserine), bleomycin (bleomycin), act-C (cactinomycin), carabicin, carminomycin (carminomycin), cardinophyllin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), Detorubicin (detorubicin), 6-phenodiazine-5-oxygen-L-nor-leucine,

come (to comprise the morpholino Doxorubicin than star (doxorubicin) more, cyano group morpholino Doxorubicin, 2-pyrroles is for Doxorubicin and deoxidation Doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycins) is such as mitomycin C, mycophenolic acid (mycophenolic acid), nogalamycin (nogalamycin), olivomycin (olivomycin), Peplomycin (peplomycin), potfiromycin, puromycin (puromycin), triferricdoxorubicin (quelamycin), rodorubicin (rodorubicin), streptonigrin (streptonigrin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), Zinostatin (zinostatin), zorubicin (zorubicin), the antimetabolite class, such as methotrexate (MTX) (methotrexate) and 5 FU 5 fluorouracil (5-FU), folacin, such as denopterin (denopterin), methotrexate (MTX) (methotrexate), pteropterin (pteropterin), Trimetrexate (trimetrexate), purine analogue, such as fludarabine (fludarabine), Ismipur (mercaptopurine), ITG (thiamiprine), thioguanine (thioguanine), pyrimidine analogue, such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (azauridine), Carmofur (carmofur), cytarabine (cytarabine), two BrdU (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), androgens, such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), Mepitiostane (mepitiostane), Testolactone (testolactone), anti-adrenal gland class, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), Trilostane (trilostane), folic acid supplement, such as folinic acid (folinic acid), aceglatone (aceglatone), aldophosphamide glucosides (aldophosphamide glycoside), amino-laevulic acid (aminolevulinic acid), eniluracil (cniluracil), amsacrine (amsacrine), bestrabucil, bisantrene (bisantrene), Edatrexate (cdatraxate), Defosfamide (defosfamide), demecolcine (demecolcine), diaziquone (diaziquone), elfornithine, Elliptinium Acetate (elliptinium acetate), Epothilones (epothilone), ethoglucid (etoglucid), gallium nitrate, hydroxyl urea (hydroxyurea), lentinan (lentinan), Lonidamine (lonidamine), maytansinoid class (maytansinoids), such as maytansine (maytansine) and ansamitocin (ansamitocin), mitoguazone (mitoguazone), mitoxantrone (mitoxantrone), Mopidamol (mopidamol), C-283 (nitracrine), Pentostatin (pentostatin), Phenamet (phenamet), THP (pirarubicin), Losoxantrone (losoxantrone), podophyllic acid (podophyllinic acid), 2-ethyl hydrazides (ethylhydrazide), procarbazine (procarbazine), polysaccharide compound (JHS Natural Products, Eugene, OR), razoxane (razoxane), rhizomycin (rhizoxin), sizofiran (sizofiran), Spirogermanium (spirogermanium), tenuazonic acid (tenuazonic acid), triethyleneiminobenzoquinone (triaziquone), 2,2 ', 2 " RA3s, trichothecin class (trichothecenes) (especially T-2 toxin, verrucarine (verrucarin) A, roridin (roridin) A and the rhzomorph (anguidin) that crawls), urethane (urethan), eldisine (vindesine), Dacarbazine (dacarbazine), mannomustine (mannomustine), dibromannitol (mitobronitol), mitolactol (mitolactol), pipobroman (pipobroman), gacytosine, cytarabine (arabinoside) (" Ara-C "), endoxan (cyclophosphamide), phosphinothioylidynetrisaziridine (thiotepa), taxoid class (taxoids), for example

Taxol (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE ^TMTaxol not containing cremophor (Cremophor), albumin transformation nano particle formulation (American Pharmaceutical Partners, Schaumberg, Illinois) and

Taxotere (doxetaxel) (

Rorer, Antony, France), Chlorambucil (chlorambucil),

gemcitabine (gemcitabine), 6-thioguanine (by ioguanine), purinethol (mercaptopurine), methotrexate (MTX) (methotrexate), platinum analogs, such as cis-platinum (cisplatin) and carboplatin (carboplatin), vincaleukoblastinum (vinblastine), platinum (platinum), Etoposide (etoposide) (VP-16), ifosfamide (ifosfamide), mitoxantrone (mitoxantrone), vincristine (vincristine), vinorelbine (vinorelbine), NSC-279836 (novantrone), Teniposide (teniposide), Edatrexate (edatrexate), daunomycin (daunomycin), aminopterin (aminopterin), Xeloda (xeloda), ibandronate (ibandronate), Irinotecan (irinotecan) (Camptosar, CPT-11) (therapeutic scheme that comprises Irinotecan and 5-FU and folinic acid), topoisomerase enzyme inhibitor RFS 2000, DFMO (DMFO), retinoic acid-like class (retinoids), such as retinoic acid (retinoic acid), capecitabine (capecitabine), combretastatin (combretastatin), folinic acid (leucovorin) (LV), oxaliplatin (oxaliplatin), comprise oxaliplatin treatment scheme (FOLFOX), PKC-α, Raf, H-Ras and EGFR (erlotinib (Tarceva for example ^TM)), the inhibitor that reduces cell proliferation, and the acceptable salt of pharmacy, acid or the derivative of any above-mentioned substance.

This definition also comprise act on regulate or inhibitory hormone to the antihormone agent of function of tumor such as anti-estrogens and selective estrogen receptor adjusting control agent class (SERM), comprise that for example tamoxifen (tamoxifen) (comprises tamoxifen), raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxytamoxifen, trioxifene (trioxifene), that Lip river former times fragrant (keoxifene), LY117018, onapristone (onapristone) and

toremifene (toremifene); Be suppressed in suprarenal gland the aromatase inhibitor of the aromatase enzyme of regulating estrogen production, such as 4 (5)-imidazoles for example, aminoglutethimide (aminoglutethimide),

magace (megestrolacetate), exemestane (exemestane), formestane (formestane), fadrozole (fadrozole),

r 83842 (vorozole),

letrozole (letrozole) and anastrozole (anastrozole); Anti-androgens, such as Drogenil (flutamide), Nilutamide (nilutamide), than Ka meter Te (bicalutamide), Leuprolide (leuprolide) and goserelin (goserelin); And troxacitabine (troxacitabine) (DOX nucleosides cytosine(Cyt) analogue); Antisense oligonucleotide, the signal that particularly suppresses to involve abnormal (abherant) cell proliferation by way of in the antisense oligonucleotide of genetic expression, such as for example PKC-α, Raf and H-Ras; Ribozyme, such as the vegf expression inhibitor (for example

nucleic acid) and the HER2 expression inhibitor; Vaccine, for example, such as the gene therapy vaccine, vaccine, vaccine and

vaccine;

rIL-2;

topoisomerase 1 inhibitor;

rmRH; Vinorelbine (Vinorelbine) and Ai Sibo mycin (Esperamicins) (seeing U.S. Patent No. 4,675,187); And pharmacologically acceptable salts, acid or the derivative of any above-mentioned substance.

Term " prodrug " refers to that for the application the time to compare the cytotoxicity of diseased cells less and can the enzymatic activation or change precursor or the derivative form of the active medicinal matter (for example small molecules) that has more active female medicine form into female medicine (parent drug).Referring to for example Wilman, " Prodrugs in CancerChemotherapy ", Biochemical Society Transactions, 14, pp.375-382,615thMeeting Belfast (1986) and Stella etc., " Prodrugs:A Chemical Approach to TargetedDrug Delivery ", Directed Drug Delivery, Borchardt etc., compile pp.247-267, HumanaPress (1985).Prodrug of the present invention includes but not limited to phosphate-containing/ester prodrug, containing sulfo-phosphate/ester prodrug, containing sulfate/ester prodrug, containing the peptide prodrug, the amino acid modified prodrug of D-, the glycosylation prodrug, containing the beta-lactam prodrug, containing the prodrug of optional substituted benzene oxygen yl acetamide or containing the prodrug of optional substituted benzene ethanamide, can be converted into and have more activity and 5-flurocytosine and other 5-FUD prodrug of the medicine of no cytotoxicity.The example that can derive the cytotoxic drug of the prodrug form of using for the present invention includes but not limited to above-described those chemotherapeutics.

" separation ", when describing each peptide species disclosed herein, mean to identify and with/certainly express its cell or the cell culture polypeptide that separates and/or reclaim.The contaminative composition of the natural surroundings of polypeptide refers to usually can disturb the material of its diagnosis or therepic use, can comprise the solute of enzyme, hormone and other oroteins character or nonprotein character.In preferred embodiments, peptide purification to (1) is enough to by using the rotary-cup type sequenator to obtain the N-end of at least 15 residues or the degree of internal amino acid sequence, or (2) reach homogeneity according to using Coomassie blue or the irreducibility of preferably silver dyeing or the SDS-PAGE under reductive condition.Since at least one composition of the natural surroundings of polypeptide can not exist, isolated polypeptide comprises the original position polypeptide in reconstitution cell so.Yet usually prepared by least one purification step by isolated polypeptide.

" separation " peptide coding nucleic acid or other peptide coding nucleic acid refer to identify and with the natural origin of peptide coding nucleic acid at least one contaminative nucleic acid molecule of associated separates usually nucleic acid molecule.The isolated polypeptide coding nucleic acid molecule is different from form or the background when occurring in nature is found it.The isolated polypeptide coding nucleic acid molecule therefore specific polypeptide encoding nucleic acid molecule when being present in n cell is had any different.For example, yet the isolated polypeptide coding nucleic acid molecule comprises the polypeptide encoding nucleic acid molecule comprised in the cell of common this polypeptide of expression, when the chromosomal localization of described nucleic acid molecule in described cell is different from its chromosomal localization in n cell.

Term " control sequence " refers to express in the specific host organism the necessary DNA sequence dna of encoding sequence be operatively connected.For example, be suitable for procaryotic control sequence and comprise promotor, optional operator gene sequence and ribosome bind site.The known genuine karyocyte utilizes promotor, polyadenylation signal and enhanser.

If one section nucleic acid and another section nucleotide sequence are in functional mutual relationship, it is " being operatively connected ".For example, if presequence (presequence) or the DNA that secretes leading (secretory leader) are expressed as the front protein (preprotein) that participates in the polypeptide secretion, the DNA of it and this polypeptide is operatively connected; If promotor or enhanser affect transcribing of encoding sequence, it and this sequence are operatively connected; Perhaps, if the position of ribosome bind site promotes translation, it and encoding sequence are operatively connected.Generally speaking, " being operatively connected " means that connected DNA sequence dna is adjacent, and in the leading situation of secretion, means adjacent and in read state.Yet enhanser needn't be adjacent.Connection can be by realizing in the connection at restriction site place easily.If there is no this type of site, according to conventional practice, use synthetic oligonucleotide adapter or joint.

As defined herein, " stringent condition " or " high stringent condition " can be defined as follows: (1) is used low ionic strength and high temperature for washing, 0.015M sodium-chlor/0.0015M Trisodium Citrate/0.1% sodium lauryl sulphate for example, 50 ℃, (2) use denaturing agent during hybridizing, such as methane amide, for example, 50% (v/v) methane amide and 0.1% bovine serum albumin(BSA)/0.1%Ficoll/0.1% polyvinylpyrrolidone/have 750mM sodium-chlor, the 50mM sodium phosphate buffer pH 6.5 of 75mM Trisodium Citrate, 42 ℃, or (3) are being used 50% methane amide, 5x SSC (0.75M NaCl, 0.075M Trisodium Citrate), 50mM sodium phosphate (pH6.8), 0.1% trisodium phosphate, 5x DenhardtShi solution, the salmon sperm DNA of supersound process (50 μ g/ml), in the solution of 0.1%SDS and 10% sulfuric acid dextran, in 42 ℃ of hybridization, spend the night, in 42 ℃, in 0.2x SSC (sodium chloride/sodium citrate), wash 10 minutes, then in 55 ℃ of high strict washings in 10 minutes of being formed by the 0.1x SSC that contains EDTA.

" per-cent (%) amino acid sequence identity " about the peptide sequence identified herein is defined as the contrast sequence and introduces where necessary breach with after obtaining largest percentage sequence identity, and not by any conservative substituting while being considered as sequence identity a part of, the percentage of the amino-acid residue identical with amino-acid residue in compared polypeptide in candidate sequence.Various ways for the contrast of measuring per-cent amino acid sequence identity purpose in can the art technology scope carries out, and for example uses the available computer software of the public, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine for measuring the suitable parameter of contrast, comprise institute's comparative sequences total length is obtained to the required any algorithm of maximum contrast.Yet for the purposes of the present invention, % amino acid sequence identity value is to use sequence to compare computer program ALIGN-2 to obtain.ALIGN-2 sequence comparison computer program is write by Genentech company, and source code (table 1) is submitted to (the US Copyright Office of U.S. Copyright Bureau together with customer documentation, Washington D.C., 20559), and with U.S. copyright registration TXU510087 register.The public can pass through Genentech company (South San Francisco, California) and obtain the ALIGN-2 program.The ALIGN2 program should be compiled in UNIX operating system, on preferred digital UNIX V4.0D, uses.The all sequences comparative parameter is by ALIGN-2 program setting and constant.

Aminoacid sequence described herein is continuous aminoacid sequence, except as otherwise noted.

For this paper the time, term " immunoadhesin " refers to the antibody molecule that the effector functions of the binding specificity of heterologous protein (" adhesin ") and immunoglobulin (Ig) constant domain is joined together.Structurally, immunoadhesin comprises aminoacid sequence with the expectation binding specificity that is different from antibody antigen identification and binding site (being " allos ") and the fusions of immunoglobulin (Ig) constant domain sequence.The adhesin of immunoadhesin molecule partly is typically the continuous amino acid sequence of the binding site that at least comprises acceptor or part (such as VEGFR or fibronectin part).Immunoglobulin (Ig) constant domain sequence in immunoadhesin can obtain from any immunoglobulin (Ig), such as IgG-1, IgG-2, IgG-3 or IgG-4 hypotype, IgA (comprising IgA-1 and IgA-2), IgE, IgD or IgM.Usually comprising the sequence selected derived from the phage display of the sequence of specific binding target thing and its merges to the peptide body (peptibody) of immunoglobulin Fc part and can be considered as immunoadhesin herein.

Term " antibody " is used with broad sense, for example clearly cover monoclonal antibody (comprising excitability, Antagonism and neutrality antibody), there is the specific antibody compositions of multi-epitope, polyclonal antibody, single-chain antibody, and antibody fragment (seeing below), as long as their specific binding natural polypeptidess and/or show biologic activity of the present invention or immunologic competence.According to an embodiment, the target protein of antibodies oligomer form, for example trimeric form.According to another embodiment, the antibodies specific conjugated protein, this combination can be subject to the inhibition of monoclonal antibody of the present invention (such as antibody of preservation of the present invention etc.)." functional fragment or the analogue " of phrase antibody refers to have with indication antibody the compound of common qualitative biologic activity.For example, the functional fragment of antibody of the present invention or analogue can be functional fragment or the analogues of energy specific binding VEGF or α 5 β 1.In one embodiment, the ability that antibody capable stops or the substantive VEGF of reduction inducing cell is bred.

" separation " antibody refers to identify and from a kind of composition of its natural surroundings separately and/or the antibody reclaimed.The contaminative composition of its natural surroundings refers to disturb the diagnosis of this antibody or the material of therepic use, can comprise the solute of enzyme, hormone and other oroteins character or nonprotein character.In preferred embodiments, by antibody purification to (1) mensuration according to the Lowry method, antibody weight surpasses 95%, most preferably weight surpasses 99%, (2) be enough to by using the rotary-cup type sequenator to obtain the N-end of at least 15 residues or the degree of internal amino acid sequence, or (3) reach homogeneity according to the SDS-PAGE under reductibility or irreducibility condition and use Coomassie blue or preferred silver dyeing.Since at least one composition of antibody natural surroundings can not exist, the antibody separated so comprises the original position antibody in reconstitution cell.Yet usually will prepare by least one purification step by the antibody of separation.

(IgM antibody is comprised of 5 basic different tetramer unit and the other polypeptide that is called the J chain the different tetramer glycoprotein that 4 basic chain antibody unit consist of two identical light chains (L) and two identical heavy chains (H), so comprises 10 antigen binding sites; And secretory IgA antibody polymerizable and forming comprises 2-5 4 basic chain units and the multivalence assemblage of J chain).In the situation of IgG, 4 chain units are about 150,000 dalton normally.Every light chain is connected with heavy chain by a covalent disulfide bonds, and two heavy chains are connected with each other by one or more disulfide linkage, and the number of disulfide linkage depends on the isotype of heavy chain.Every heavy chain and light chain also have disulphide bridges in the chain of interval rule.Every heavy chain has a variable domain (V at the N-end _h), be then three (for α and γ chains) or four (for μ and ε isotype) constant domain (C _h).Every light chain has a variable domain (V at the N-end _l), be then a constant domain (C of its other end _l).V _lwith V _harranged together, and C _lwith heavy chain the first constant domain (C _h1) arranged together.Think that specific amino-acid residue forms interface between light chain and variable region of heavy chain.A V _hwith a V _lmatch together and form an antigen binding site.About structure and the character of different classes of antibody, referring to for example " Basicand Clinical Immunology) ", the 8th edition, Daniel P. Stites, Abba I.Terr and Tristram G.Parslow (volume), Appleton & Lange, Norwalk, CT, 1994, the 71 pages and the 6th chapter.

According to its constant domain aminoacid sequence, can be included into a kind of in two kinds of completely different types from the L chain of any invertebrate species, be called card handkerchief (κ) and lambda (λ).According to its heavy chain constant domain (C _h) aminoacid sequence, immunoglobulin (Ig) can be included into different classifications or isotype.Five immunoglobulin like protein: IgA, IgD, IgE, IgG and IgM are arranged, there is respectively the heavy chain that is called α, δ, ε, γ and μ.According to C _hthe less difference of sequence and function, γ and α class can be further divided into subclass, and for example the mankind express following subclass: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

Term " variable " refers to some section in variable domain sequence difference truth widely between antibody.V structural domain mediation antigen combination also limits the specificity of specific antibodies to its specific antigen.Yet variability not is uniformly distributed in 110 amino acid that variable domain is crossed over.In fact, the V district is by 15-30 amino acid, and being called the section relatively do not made a variation of framework region (FR) and each length that framework region is separated is 9-12 amino acid, and the shorter zone that is called the extreme variation of " hypervariable region " forms.Each self-contained four FR of the variable domain of natural heavy chain and light chain, they take the beta-pleated sheet conformation mostly, and three hypervariable regions that form a beta-pleated sheet structure part by the formation loop connecting and in some situation connect.What the hypervariable region in every chain approached by FR very much keeps together, and facilitate the formation of the antigen binding site of antibody (referring to Kabat et al. with together with the hypervariable region of another chain, Sequences of Proteins of ImmunologicalInterest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD (1991)).Constant domain is not participated in the combination of antibody and antigen directly, but shows multiple effector functions, such as the participation of antibody in the cytotoxicity (ADCC) of antibody dependent cellular.

Term " hypervariable region " refers to the amino-acid residue of the responsible antigen combination of antibody for this paper the time.Hypervariable region for example generally comprises, from the amino-acid residue of " complementary determining region " or " CDR " (V _lin residue 24-34 (L1), 50-56 (L2) and 89-97 (L3) near and V _hin residue 31-35B (H1), 50-65 (H2) and 95-102 (H3) near (in one embodiment, H1 is near residue 31-35); Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed.Public HealthService, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from " hypermutation ring " (V for example _lin residue 26-32 (L1), 50-52 (L2) and 91-96 (L3) and V _hin residue 26-32 (H1), 53-55 (H2) and 96-101 (H3); Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)).

Term " monoclonal antibody " refers to that for this paper the time each antibody individuality that forms colony is identical from a group antibody that the antibody of homogeneity obtains basically, except can be with indivisible possible natural the existence mutant form existed.Monoclonal antibody is high degree of specificity, for single antigenic site.In addition, from the polyclonal antibody prepared product difference comprised for the different antibodies of different determinants (epi-position), every kind of monoclonal antibody is for the single determinant on antigen.Outside their specificity, the advantage of monoclonal antibody is that they can be synthetic in the situation that is not subject to other antibody pollution.Modifier " mono-clonal " should not be construed as and requires to generate antibody by any ad hoc approach.For example, can be used for monoclonal antibody of the present invention can prepare by the hybridoma method of being put down in writing by Nature 256:495 (1975) such as Kohler at first, perhaps can in bacterium, eucaryon animal or plant cell, prepare (referring to for example U.S. Patent No. 4 by recombinant DNA method, 816,567)." monoclonal antibody " also can be used such as Nature 352:624-628 (1991) such as Clackson; The J.Mol.Biol.222:581-597 such as Marks (1991) separate from phage antibody library with the technology of putting down in writing in embodiment hereinafter.

Monoclonal antibody comprises " chimeric " antibody in this article, the wherein part of heavy chain and/or light chain and the identical or homology of the corresponding sequence in the antibody of specific antibodies classification or subclass derived from the Special Thing species or genus, and the remainder of chain with derived from another species or belong to the identical or homology of corresponding sequence in the antibody of another antibody isotype or subclass, and the fragment of this antibody-like, as long as they show biologic activity of the present invention (referring to U.S. Patent No. 4,816,567; Morrison et al., Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984)).Interested chimeric antibody comprises and comprising derived from the variable domain antigen binding sequence of non-human primate (such as Old World monkey class (Old World Monkey), ape etc.) and " primatesization " antibody of human constant region sequence herein.

" complete antibody " refers to comprise antigen binding site and C _lheavy chain constant domain C at least _h1, C _h2 and C _h3 antibody.Constant domain can be native sequences constant domain (for example naive sequence constant domain) or its aminoacid sequence variant.Preferably, complete antibody has one or more effector functions.

The part that " antibody fragment " comprises complete antibody, the preferably combination of the antigen of complete antibody or variable region.The example of antibody fragment comprises Fab, Fab ', F (ab ') ₂with the Fv fragment; Double antibody; (referring to U.S. Patent No. 5,641,870, embodiment 2 for linear antibody; Zapata et al., Protein Eng.8 (10): 1057-1062 (1995)); The single-chain antibody molecule; And the multi-specificity antibody formed by antibody fragment.

Statement " linear antibody " refers generally to Zapata et al., Protein Eng, 8 (10): the antibody described in 1057-1062 (1995).In brief, the Fd section (VH-CH1-VH-CH1) that these antibody comprise pair of series, this section forms a pair of antigen binding domain with together with complementary light chain polypeptide.Linear antibody can be dual specific, or monospecific.

Produce two identical Fabs with papain digestion antibody, be called " Fab " fragment, and remnants " Fc " fragment, its title has reflected that it is easy to the ability of crystallization.The Fab fragment is by the variable domain (V of a complete light chain and a heavy chain _h) and the first constant domain (C _h1) form.Each Fab fragment is for antigen in conjunction with being unit price, and it has an antigen binding site.Pepsin antibody produces a larger F (ab ') ₂fragment, it is equivalent to roughly two Fab fragments connected by disulfide linkage, has divalence antigen-binding activity and still can crosslinked antigen.Fab ' fragment is because of at C _hthe C-terminal of 1 structural domain has increased the minority residue and is different with the Fab fragment, comprises the one or more halfcystines from antibody hinge region.Fab '-SH is herein to appellation that wherein the constant domain cysteine residues carries the Fab ' of a free sulphur alcohol radical.F (ab ') ₂antibody fragment is to generate as paired Fab ' fragment at first, between Fab ' fragment, has hinge cysteine.Also know other chemical coupling form of antibody fragment.

The C-terminal part that the Fc fragment comprises two heavy chains that keep together by disulfide linkage.The part that the Fc acceptor (FcR) that sequence decision ，Gai district in the effector functions Shi You Fc district of antibody still is subject to finding on some cell type is identified.

" Fv " is the minimum antibody fragment that comprises complete antigen recognition and binding site.This fragment is comprised of tight a, variable region of heavy chain of non-covalent combination and the dimer of a variable region of light chain.Give out six hypermutation rings (each 3 rings of heavy chain and light chain) from this two structural domains folding, contribute the amino-acid residue of antigen combination and give antibody with antigen-binding specificity.Yet, even single variable domain (or only comprising half Fv to three CDR of antigen-specific) also has the ability of identification and conjugated antigen, although avidity is lower than complete binding site.

" scFv ", also can be abbreviated as " sFv " or " scFv ", is to comprise the antibody V that connects into a polypeptide chain _hand V _lthe antibody fragment of structural domain.Preferably, the sFv polypeptide is at V _hand V _lalso comprise peptide linker between structural domain, make sFv form the structure of antigen in conjunction with expectation.About the summary of sFv referring to Pluckthun, in " The Pharmacology of Monoclonal Antibodies ", vol.113, Rosenburg and Moore compile, Springer-Verlag, New York, pp.269-315,1994; Borrebaeck 1995, see below.

Term " double antibody " refers to by V _hand V _lbetween structural domain, use short circuit head (an about 5-10 residue) to build sFv fragment (seeing the preceding paragraph) and the small-sized antibody fragment of preparation, because joint is short, make the V structural domain carry out interchain but not pairing in chain causes the divalence fragment, there is the fragment of two antigen binding sites.The dual specific double antibody is the heterodimer of two " intersection " sFv fragments, and wherein the VH of two kinds of antibody and VL structural domain are present on different polypeptide chains.Double antibody is more complete is described in for example EP 404,097; WO93/11161; Hollinger et al., Proc.Natl.Acad.Sci.USA, 90:6444-6448 (1993).

" humanization " form of inhuman (for example rodents) antibody refers to that bottom line comprises the chimeric antibody derived from non-human antibody's sequence.Largely, humanized antibody refers to the immunoglobulin (Ig) that the hypervariable region residue in human normal immunoglobulin (receptor antibody) is replaced with the hypervariable region residue of inhuman species (donor antibody) such as mouse, rat, rabbit or non-human primates with expectation antibodies specific, avidity and ability.In some situation, the framework region of human normal immunoglobulin (FR) residue is replaced with corresponding inhuman residue.In addition, humanized antibody can be included in receptor antibody or donor antibody does not have the residue of finding.Carrying out these modifications is in order further to improve the performance of antibody.Usually, humanized antibody comprise at least one, common two whole following variable domains basically, wherein all or basically all hypermutation rings corresponding to the hypermutation ring of non-human immunoglobulin, and all or basically all FR are FR of human normal immunoglobulin sequence.Humanized antibody optionally also comprises at least part of constant region for immunoglobulin (Fc), the normally constant region of human normal immunoglobulin.More details are referring to Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988); Presta, Curr.Op.Struct.Biol.2:593-596 (1992).

" species dependency antibody " refers to that the antigen from the first mammalian species is had and is better than the antibody of this antigen from the binding affinity of the homologue of the second mammalian species.Usually, species dependency antibody " specific binding " mankind antigen (has and is no more than approximately 1 x 10 ^-7m, preferably be no more than approximately 1 x 10 ^-8m, be most preferably not exceeding approximately 1 x 10 ^-9the binding affinity of M (Kd)), but to this antigen from the homologue of the second non-human mammal species have than its to the binding affinity of mankind's antigen a little less than at least about 50 times, or at least about 500 times, or at least about the binding affinity of 1000 times.Species dependency antibody can be all kinds antibody defined above, but preferred humanized antibody or people's antibody.

In this type of embodiment, according to fluorescence-activated cell sorting (FACS), analyze or the mensuration of radioimmunoprecipitation (RIA), polypeptide, antibody, antagonist or composition will be less than this polypeptide, antibody, antagonist or composition to approximately 10% of the combination of its particular target thing protein in conjunction with the degree of " non-target thing " protein.Combination for polypeptide, antibody, antagonist or composition to target molecule, the epi-position on term " specific combination " or " specific binding " specific polypeptide or specific polypeptide target thing or its " special " meaned to measurable non-specific interactional combination that is different from.Specific combination can by for example measure molecule in conjunction with and with contrast molecule in conjunction with relatively measuring, described contrast molecule is structural similitude but less than in conjunction with active molecule normally.For example, specific combination can be measured by the competition with contrasting molecule, described contrast molecule and target phase seemingly, excessive unmarked target thing for example.In this case, if be subject to the competitive inhibition of excessive unmarked target thing through the combination of labels targets thing and probe, indicate specific combination.Epi-position on term " specific combination " or " specific binding " specific polypeptide or specific polypeptide target thing or can be at least about 10 by for example Kd to the target thing to its " special " for this paper the time ^-4m, or at least about 10 ^-5m, or at least about 10 ^-6m, or at least about 10 ^-7m, or at least about 10 ^-8m, or at least about 10 ^-9m, or at least about 10 ^-10m, or at least about 10 ^-11m, or at least about 10- ¹²m or larger molecule represent.In one embodiment, term " specific combination " refers to such combination, and wherein molecule is in conjunction with the epi-position on specific polypeptide or specific polypeptide, and basically not in conjunction with any other polypeptide or polypeptide epitope.

Antibody " effector functions " refers to the biologic activity that those are attributable to antibody Fc district (native sequences Fc district or aminoacid sequence variant Fc district) and change with antibody isotype.The example of antibody mediated effect device function comprises: C1q combination and CDC; The Fc receptors bind; The cytotoxicity (ADCC) of antibody dependent cellular mediation; Phagolysis; Cell surface receptor is lowered; With the B cell activation.

The identical aminoacid sequence of aminoacid sequence that " native sequences Fc district " comprises the Fc district found with occurring in nature.The example of Fc sequence is recorded in such as but not limited to Kabat etc., " Sequences ofImmunological Interest ", the 5th edition, Public Health Service, National Institutes ofHealth, Bethesda, Md, 1991.

" variant Fc regions " comprises due at least one place " amino acid modified " defined herein the aminoacid sequence different with native sequences Fc district.Preferably, variant Fc regions has with native sequences Fc district or with the Fc district of parent's polypeptide compares at least one place amino acid replacement, for example in native sequences Fc district or in the Fc district of parent's polypeptide, there is approximately 1 place to about 10 place's amino acid replacements, preferably approximately 1 place to about 5 place's amino acid replacements.In one embodiment, variant Fc regions will have the homology at least about 80% with native sequences Fc district in this article, the homology at least about 85%, the homology at least about 90%, the homology at least about 95% or at least about 99% homology.According to another embodiment, variant Fc regions will have the homology at least about 80% with the Fc district of parent's polypeptide in this article, the homology at least about 85%, the homology at least about 90%, the homology at least about 95% or at least about 99% homology.

Be defined as contrast sequence and will any conservative alternative while being considered as sequence identity a part of about " per-cent (%) amino acid sequence identity " or " homology " of the polypeptide identified and antibody sequence herein, the percentage of the amino-acid residue identical with amino-acid residue in compared polypeptide in candidate sequence.Various ways for the contrast of measuring per-cent amino acid sequence identity purpose in can the art technology scope carries out, and for example uses the available computer software of the public, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine for measuring the suitable parameter of contrast, comprise institute's comparative sequences total length is obtained to the required any algorithm of maximum contrast.Yet for the purposes of the present invention, % amino acid sequence identity value is to use sequence to compare computer program ALIGN-2 to obtain.ALIGN-2 sequence comparison computer program is write by Genentech company, and source code is submitted to (the US Copyright Office of U.S. Copyright Bureau together with customer documentation, Washington D.C., 20559), and register with U.S. copyright registration TXU510087.The public can pass through Genentech company (South San Francisco, California) and obtain the ALIGN-2 program.The ALIGN2 program should be compiled in UNIX operating system, on preferred digital UNIX V4.0D, uses.The all sequences comparative parameter is by ALIGN-2 program setting and constant.

Term “Han Fc district polypeptide " refer to the polypeptide that comprises the Fc district, such as antibody or immunoadhesin (definition sees below).The C-end Methionin in Fc district (according to the residue 447 of EU numbering system) can be eliminated, for example, in the process of purified polypeptide or by the nucleic acid of modified recombinant coded polypeptide.Therefore, the composition that comprises the polypeptide (comprising antibody) with Fc district according to the present invention can comprise the polypeptide colony that all K447 residues all are eliminated, polypeptide colony or the polypeptide with K447 residue and the mixture that there is no the polypeptide of K447 residue that none K447 residue is eliminated.

Run through this specification sheets and claims, the Kabat numbering system is generally used during the residue in mentioning variable domain (being approximately light chain residue 1-107 and heavy chain residue 1-113) (such as Kabat etc., " Sequencesof Immunological Interest ", the 5th edition, Public Health Service, National Institutes ofHealth, Bethesda, Md, 1991).During " EU numbering system " or " EU index " general residue in mentioning immunoglobulin heavy chain constant region, use (such as Kabat etc., " Sequences of Proteinsof Immunological Interest ", the 5th edition, Public Health Service, National Institutes ofHealth, Bethesda, MD, in 1991, the EU index of report, clearly be collected herein by reference).Unless be otherwise noted herein, mentioned that the residue numbering in antibody variable domains refers to the residue numbering according to the Kabat numbering system.Unless be otherwise noted herein, mentioned that the residue numbering in the antibody constant domain refers to the residue numbering according to the EU numbering system.

Term " Fc acceptor " or " FcR " are for describing the acceptor in binding antibody Fc district.In one embodiment, FcR of the present invention refers to comprise the acceptor of Fc γ RI, Fc γ RII and Fc γ RIII subclass in conjunction with the FcR of IgG antibody (γ acceptor), comprises allelic variant and the alternative splicing form of these acceptors.Fc γ RII acceptor comprises Fc γ RIIA (" activated receptor ") and Fc γ RIIB (" inhibition acceptor "), and they have similar aminoacid sequence, and difference mainly is its cytoplasmic structure territory.Activated receptor Fc γ RIIA comprises the activation motif (ITAM) of immunity receptor based on tyrosine in its cytoplasmic structure territory.Suppress acceptor Fc γ RIIB and comprise the inhibition motif (ITIM) of immunity receptor based on tyrosine (referring to summary in its cytoplasmic structure territory , Annu.Rev.Immunol.15:203-234 (1997)).This term comprises allotype (allotype), such as Fc γ RIIIa allotype: Fc γ RIIIA-Phe158, Fc γ RIIIA-Val158, Fc γ RIIA-R131 and/or Fc γ RIIA-H131.The summary of FcR is referring to Ravetch and Kinet, Annu.Rev.Immunol.9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); De Haas et al., J.Lab.Clin.Med.126:330-41 (1995).Other FcR contained in this article in term " FcR ", comprises what will identify those futures.This term also comprises newborn infant's acceptor, FcRn, and it is responsible for Maternal immunoglobulin G is transferred to fetus (Guyer et al., J.Immunol.117:587 (1976); Kim et al., J.Immunol.24:249 (1994)).

Term " FcRn " refers to neonatal Fc receptor (FcRn).FcRn is structurally similar to ajor histocompatibility mixture (MHC), α-chain and the B2M of non-covalent combination, consists of.The summary of the several functions of neonatal Fc receptor FcRn is shown in Ghetie and Ward (2000) Annu.Rev.Immunol.18,739-766.FcRn plays a role from the passive young baby of being delivered to of parent with in regulating Serological IgG level at immunoglobulin IgG.FcRn can play the effect of remedying acceptor, cell neutralization pass through Cell binding and transhipment complete form by pinocytosis IgG, and save the degradation pathway that they avoid acquiescence.

WO00/42072 (Presta) and Shields et al., J.Biol.Chem.9 (2): 6591-6604 (2001) put down in writing to FcR in conjunction with the antibody variants being improved or eliminate.The content of these publications clearly is collected herein by reference.

" the CH1 structural domain " in human IgG Fc district (also referred to as " C1 " or " H1 " structural domain) extends to about amino acid 215 (EU numbering system) from about amino acid/11 18 usually.

" hinge area " is normally defined from the section of human IgG1's Glu216 to Pro230 (Burton, Molec.Immunol.22:161-206 (1985)).The hinge area of other IgG isotype can be by being placed in first and the cysteine residues that last forms S-S key between heavy chain same position and contrasting with the IgG1 sequence.

" the rudimentary hinge area " in Fc district is normally defined the residue 233-239 in the one section residue ，Ji Fc district that is close to hinge area C-end.In previous report, FcR is in conjunction with amino-acid residue in the rudimentary hinge area of generally giving the credit to IgG Fc district.

" the CH2 structural domain " in human IgG Fc district (also referred to as " C2 " or " H2 " structural domain) extends to about amino acid 340 from about amino acid 231 usually.The unique distinction of CH2 structural domain is that it closely not match with another structural domain.But the carbohydrate chain of the branch that has two N-to connect is between two CH2 structural domains of complete natural IgG molecule.Infer that carbohydrate may provide substituting of structural domain-structural domain pairing and contribute to stablize the CH2 structural domain.Burton，Molec.Immunol.22：161-206(1985)。

One section residue that " CH3 structural domain " (also referred to as " C3 " or " H3 " structural domain) comprises CH2 domain C-end in the Fc district (from about amino-acid residue 341 of antibody sequence to C-terminal, the amino-acid residue 446 or 447 of IgG normally).

" functional Fc district " has " effector functions " in native sequences Fc district.Exemplary " effector functions " comprises cytotoxicity (ADCC), phagolysis, the cell surface receptor (B-cell receptor for example of Clq combination, CDC, Fc receptors bind, antibody dependent cellular mediation; BCR) lower etc.This type of effector functions general requirement Fc district for example, combines with binding domains (antibody variable domains), and can assess by the many measure method, for example disclosed herein.

" Clq " refers to comprise the polypeptide for the site of immunoglobulin fc region combination.Clq is that Clr forms mixture Cl together with Cls with two kinds of serine proteases, i.e. the first component of CDC (CDC) approach.People Clq can be purchased from for example Quidel, San Diego, CA.

Term " binding domains " refers to can be in conjunction with the zone of another molecule in polypeptide.In the situation of FcR, binding domains can comprise the part of for example, being responsible for the Fc combination in its polypeptide chain (its α chain).A kind of useful binding domains is the ectodomain of FcR α chain.

There is FcR binding affinity or ADCC active " changes " the antibody of variation IgG Fc or peptide body refer to compare with parent's polypeptide or with the polypeptide that comprises native sequences Fc district FcR and be combined antibody or the peptide body that activity (for example Fc γ R or FcRn) and/or ADCC activity be enhanced or weaken.The variation Fc of " show to FcR in conjunction with increasing " for example, with the avidity higher than parent's polypeptide or native sequences IgG Fc (lower apparent Kd or IC50 value) in conjunction with at least one FcR.According to some embodiments, the enhancing of comparing combination with parent's polypeptide is approximately 3 times, and preferably approximately 5 times, 10 times, 25 times, 50 times, 60 times, 100 times, 150 times, 200 times until 500 times, or in conjunction with improving approximately 25% to 1000%.The polypeptide variants of " show to FcR in conjunction with reducing " for example, with the avidity lower than parent's polypeptide (higher apparent Kd or higher IC50 value) in conjunction with at least one FcR.Compare weakening of combination with parent's polypeptide and can be approximately 40% or more reductions of combination.

" antibody dependent cellular mediation cytotoxicity " or " ADCC " refers to wherein be attached to the target cell that for example, secretor type Ig on the upper Fc acceptor (FcR) existed of some cytotoxic cell (NK cell (NK) cell, neutrophilic granulocyte and scavenger cell) makes these cytotoxic effect cells can specific binding carry antigen, the cytotoxicity form of killing subsequently target cell with cytotoxin.This antibody " arms " is cytotoxic cell (arm), and is that this type of lethal effect definitely requires.The main cell of mediation ADCC, the NK cell, only express Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.Ravetch and Kinet, Annu.Rev.Immunol.9:457-92 (1991) the 464th page table 3 has been summed up the FcR on the hematopoietic cell and has been expressed.In order to assess the ADCC activity of molecules of interest, can carry out external ADCC assay method, such as U.S. Patent No. 5,500,362 or 5,821,337 or hereinafter put down in writing in embodiment.The effector cell who can be used for this type of assay method comprises peripheral blood mononuclear cell (PBMC) and NK cell (NK) cell.Perhaps/in addition, can assess in vivo the ADCC activity of molecules of interest, for example, in animal model, such as Clynes et al., disclosed in PNAS (USA) 95:652-656 (1998).

Comprise " ADCC that shows enhancing " or refer at the polypeptide with variant Fc regions and there is the amount of polypeptide (or parent's polypeptide) in mensuration in the wild-type Fc district polypeptide of substantive more effective mediation ADCC in vitro or in vivo when substantially the same than the polypeptide of the variant Fc regions of the cytotoxicity (ADCC) of the polypeptide with wild-type IgG Fc or the mediation of the more effective mediate antibody dependent cell of parent's polypeptide when having people effector cell.Generally speaking, this type of variant is identified by external ADCC assay method presently disclosed, but is for example contained, for measuring other assay method or the method for ADCC activity, in animal model.In one embodiment, preferred variant is more effective than wild-type Fc (or parent's polypeptide) aspect mediation ADCC, and approximately 5 times to approximately 100 times, for example approximately 25 times to approximately 50 times.

" CDC " or " CDC " while referring to have complement to the dissolving of target cell.The activation of CCP is that the antibody (suitable subclass) that closes the combination of associated antigen institute in conjunction with it by complement system the first component (C1q) is initial.In order to assess complement activation, can carry out the CDC assay method, for example, as Gazzano-Santoro et al., in J.Immunol.Methods 202:163 (1996), put down in writing.

There is the Fc region amino acid sequence of change and the polypeptide of C1q binding ability enhancing or that weaken and be recorded in U.S. Patent No. 6,194,551B1 and WO99/51642.The content of these patent publications clearly is collected herein by reference.Also can be referring to Idusogie et al., J.Immunol.164:4178-4184 (2000).

The white corpuscle that " people effector cell " refers to express one or more FcR and exercise effector functions.According to an embodiment, this cell is at least expressed Fc γ RIII and is exercised the ADCC effector functions.The example of the human leukocyte of mediation ADCC comprises peripheral blood mononuclear cell (PBMC), NK cell (NK) cell, monocyte, cytotoxic T cell and neutrophilic granulocyte, preferably PBMC and NK cell.The effector cell can separate from its natural origin, for example as described herein from blood or PBMC separation.

Be known (referring to for example Ghetie 1997, Hinton 2004) for the method for measuring the FcRn combination, and be recorded in hereinafter embodiment.People FcRn high-affinity Binding peptide can be in the transgenic mice of for example expressing people FcRn or transfected with human clone to the Binding in vivo of people FcRn and serum half-life, or measure in the primate of having used Fc variation polypeptide.In one embodiment, particularly, anti-alpha 5 beta 1 antibody of the present invention with variation IgG Fc shows the binding affinity raise than the polypeptide with wild-type IgG Fc, raises at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 100 times, at least 125 times, at least 150 times.In a specific embodiment, the binding affinity of people FcRn has been improved to approximately 170 times.

About the binding affinity to FcRn, in one embodiment, the EC50 of polypeptide or apparent Kd (at pH6.0) are less than 1uM, are more preferably less than or equal 100nM, are more preferably less than or equal 10nM.In one embodiment, about raise to Fc γ RIII (F158; Be the low-affinity isotype) binding affinity, EC50 or apparent Kd are less than or equal to 10nM, and to Fc γ RIII (V158; The high-affinity isotype), EC50 or apparent Kd are less than or equal to 3nM.According to another embodiment, if test antibody and control antibodies binding curve for example, at the ratio of the absorbancy numerical value of midpoint (A _{450nm (antibody)}/ A _{450nm (non-antibody)}) be less than or equal to 40%, antibody to the Fc acceptor for example, in conjunction with respect to control antibodies ( antibody) reduction can be thought significant with respect to control antibodies.According to another embodiment, if test antibody and control antibodies binding curve for example, at the ratio of the absorbancy numerical value of midpoint (A _{450nm (antibody)}/ A _{450nm (control antibodies)}) be more than or equal to 125%, antibody to the Fc acceptor for example, in conjunction with respect to control antibodies ( antibody) rising can be thought significant with respect to control antibodies.Referring to for example embodiment 16.

" parent's polypeptide " or " parental antibody " refer to comprise polypeptide or the antibody that the aminoacid sequence that produces variation polypeptide or antibody and described variation polypeptide or antibody compare with it.Be typically, parent's polypeptide or parental antibody lack a place or many places Fc district disclosed herein and modify and compare different aspect effector functions with polypeptide variants disclosed herein.Parent's polypeptide can comprise native sequences Fc district or have the amino acid sequence modifications of being pre-existing in Fc district of (such as adding, delete and/or substituting).

Antibody of the present invention can be derived from phage display.For this paper the time, " ”Huo“ storehouse, library " refers to numerous antibody or antibody fragment sequence, or the nucleic acid of these sequences of encoding, and these sequences are different in the variant amino acid combined aspects that imports these sequences according to the inventive method.

" phage display " is that a kind of polypeptide that will make a variation is illustrated in for example, technology on phage (filobactivirus) particle surface as the fusion rotein with coat protein at least a portion.The effectiveness of phage display is can be to the large-scale library of randomization protein variant fast and the truth of those sequences that can be combined with target antigen with high-affinity of efficient separation.The displaying of peptides and proteins library on phage screens for the polypeptide to millions of the polypeptide that those have the specific binding characteristic.Polyvalent phage shows that method is for showing little random peptide and small protein, and it merges to realize by the gene III with filobactivirus or gene VIII.Wells and Lowman, Curr.Opin.Struct.Biol.3:355-362 (1992), and the reference of wherein quoting.In monovalent phages is showed, by protein or peptide library and gene III or one meromixis, and when having wild type gene III albumen with low expression level, make the fusion rotein of a copy of phage particle displaying or do not show fusion rotein.Affinity effect (avidity effect) has obtained reduction with respect to polyvalent phage, makes the ligand affinity of sorting based on inherent, and uses phagemid vector, and this has simplified the DNA operation.Lowman and Wells，Methods：A companion toMethods in Enzymology 3：205-0216(1991)。

" phagemid " is for example, plasmid vector with phage gene interval of bacterium replication orgin (Co1E1) and a copy.Phagemid can utilize any known phage, comprises filobactivirus and lambdoid phages.The mark selected that plasmid generally also comprises antibiotics resistance.The DNA section that is cloned into these carriers can equally increase by the picture element grain.When providing to the cell hold these carriers to generate the necessary all genes of phage particle, the replication mode of plasmid becomes rolling-circle replication, with the copy of a chain generating plasmid DNA, and the packing phage particle.Phagemid can form infectious or non-infectious phage particle.This term comprises such phagemid, and they comprise bacteriophage coat protein gene or its fragment that connects into the gene fusion thing with the heterologous polypeptide gene, make heterologous polypeptide be illustrated on the surface of phage particle.

Term " phage vector " refers to the double-stranded rf phage that comprises heterologous gene and can copy.Phage vector has the phage replication starting point of allowing that phage replication and phage particle form.Phage is filobactivirus preferably, such as M13, f1, fd, Pf3 phage or derivatives thereof, or lambdoid phages, such as λ, 21, ,

, the or derivatives thereofs such as 82,424,434.

Covalent modification to polypeptide such as peptide body, immunoadhesin, antibody and small peptide comprises within the scope of the invention.The covalent modification of one type comprises that the target amino acid residue that makes polypeptide reacts with organic derivatization reagent, and this organic derivatization reagent can react with the selected side chain of polypeptide or N-or C-terminal residue.Can be used for for example making polypeptide and water-fast supported matrix or surface-crosslinked with the difunctional dose of derivatize carried out, with the method for antibody purification, vice versa.Linking agent commonly used for example comprises 1; the ester that two (the diazonium ethanoyl)-2-phenylethanes of 1-, glutaraldehyde, N-hydroxy-succinamide ester for example form with the 4-azidosalicylic acid, with difunctional imido-ester, comprise that two succinimido esters are such as 3; 3 '-dithio two (succinimido-propionic esters), difunctional maleimide be such as two-N-maleimide-1, the 8-octane and such as the p-azidophenyl of methyl-3-[() dithio] reagent such as propionyl imidoether.

Other modification comprise glutaminyl and asparaginyl residue respectively deacylated tRNA amine be glutamy and aspartoyl residue; the hydroxylation of proline(Pro) and Methionin; the phosphorylation of the hydroxyl of seryl or threonyl residue; alpha-amino (the T.E.Creighton that methylates of Methionin, arginine and Histidine side chain; Proteins:Structure and Molecular Properties, W.H.Freeman & Co., San Francisco, pp.79-86 (1983)), the acetylize of N-terminal amine, and the amidation of any C-terminal carboxyl(group).

Other modification comprise by toxin conjugated to antagonist such as maytenin and maytansinoid class, calicheamicin and other cytotoxic agent.

Another kind of covalent modification to polypeptide comprises, with United States Patent(USP) Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337 described modes, be connected one of polymkeric substance of polypeptide and multiple nonprotein character, for example polyoxyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene (polyoxyalkylene).

If favourable, can also modify polypeptide of the present invention in the mode that forms chimeric molecule, described chimeric molecule comprises the polypeptide (for example immunoadhesin or peptide body) merged with another kind of heterologous polypeptide or aminoacid sequence.

In one embodiment, this type of chimeric molecule comprises the fusions of polypeptide and protein transduction domains, described protein transduction domains by polypeptide target to being delivered to Various Tissues, be exactly through hemato encephalic barrier in particular, wherein use for example protein transduction domains of human immunodeficiency virus TAT albumen (Schwarze et al., 1999, Science 285:1569-72).

In another embodiment, this type of chimeric molecule comprises the fusions with the polypeptide of label polypeptide, and described label polypeptide provides the epi-position of the alternative combination of anti-tag antibody.Epitope tag is usually located at amino or the C-terminal of polypeptide.This type of existence with the polypeptide of epitope tag form can use the antibody for described label polypeptide to detect.And the providing of epitope tag makes polypeptide be easy to carry out purifying with anti-tag antibody or another kind of affinity matrix in conjunction with described epitope tag by affinity purification.Multiple label polypeptide and separately antibody be known in the art.Example comprises polyhistidine (poly-his) or many-HIS-GLY (poly-his-gly) label; Influenza HA label polypeptide and antibody 12CA5 thereof (Field et al., Mol.Cell.Biol.8:2159-2165 (1988)); C-myc label and antibody 8F9,3C7,6E10, G4, B7 and 9E10 (Evan et al., Molecular and Cellular Biology 5:3610-3616 (1985)); And HSV gD (gD) label and antibody (Paborsky et al., Protein Engineering3 (6): 547-553 (1990)) thereof.Other label polypeptide comprises Flag peptide (Hopp et al., BioTechnology 6:1204-1210 (1988)); KT3 epitope peptide (Martin et al., Science 255:192-194 (1992)); Alpha-tubulin epitope peptide (Skinner et al., J.Biol.Chem.266:15163-15166 (1991)); And T7 gene 10 protein peptide tags (Lutz-Freyermuth et al., Proc.Natl.Acad.Sci.USA 87:6393-6397 (1990)).

In an alternative embodiment, chimeric molecule can comprise the fusions of polypeptide and immunoglobulin (Ig) or immunoglobulin (Ig) specific region.For example, for the chimeric molecule (" immunoadhesin ") of bivalent form, this type of fusions can be the fusion with IgG molecule Fc district.Ig fusions of the present invention comprises the residue 94-243, the residue 33-53 that roughly comprise or only comprise the people or the residue 33-52 polypeptide with displacement intramolecular at least one variable region of Ig.In an especially preferred embodiment, the hinge that the immunoglobulin (Ig) fusions comprises the IgG1 molecule, CH2 and CH3, or hinge, CH1, CH2He CH3 district.Preparation about the immunoglobulin (Ig) fusions also can be referring to the U.S. Patent No. 5,428,130 of bulletin on June 27 nineteen ninety-five.

The invention provides for suppressing or the method and composition of the growth of cancer cells of the tumor growth of prevention of recurrence or recurrence.In various embodiments, cancer refers to the tumor growth of recurrence or the growth of cancer cells of recurrence, and wherein the number of cancer cells does not significantly reduce, perhaps increased, perhaps gross tumor volume does not significantly dwindle, or has increased, or cancer cells volume or number failed further to dwindle/reduce.Determine whether cancer cells is that the tumor growth of recurrence or the growth of cancer cells of recurrence can carry out in vivo or in vitro by this area any method that treatment is known cancer cells validity about mensuration.It is an example of the tumor growth of recurrence that antagonism VEGF treatment has the tumour of resistance.

" significant quantity " of polypeptide disclosed herein, antibody, antagonist or composition refers to be enough to realize the amount of the purpose of clear." significant quantity " can by rule of thumb and, with currently known methods, the purpose of contact regulation be determined.

Term " treatment significant quantity " refers in Mammals (aka patient) amount of antibody of the present invention, polypeptide or the antagonist of effective " treatment " disease or illness.In the situation of cancer, the treatment significant quantity of medicine can reduce the cancer cells number; Dwindle gross tumor volume or weight; Suppress (slowing down to a certain degree, preferably stop) cancer cell infiltration in peripheral organs; Suppress (slowing down to a certain degree, preferably stop) metastases; Inhibition tumor growth to a certain degree; And/or to a certain degree alleviate one or more symptoms with related to cancer.With regard to the degree that can prevent to have growth of cancer cells now and/or kill existing cancer cells with regard to medicine, it can be the cell inhibition and/or Cytotoxic.In one embodiment, the treatment significant quantity is the growth-inhibiting amount.In another embodiment, the treatment significant quantity is the amount that extends patient's survival.In another embodiment, the treatment significant quantity is to improve the amount of patient's progresson free survival.

In the situation of wound healing, term " significant quantity " or " treatment significant quantity " refer to that medicine effectively accelerates or improve the amount of the wound healing in the experimenter.Therapeutic dose refers to the patient is shown the dosage of result for the treatment of, and inferior therapeutic dose refers to treated patient is not shown the dosage of result for the treatment of.

" chronic wounds " refers to the wound of disunion.Referring to for example Lazarus et al., Definitions andguidelines for assessment of wounds and evaluation of healing, Arch.Dermatol.130:489-93 (1994).Chronic wounds includes but not limited to such as ulcer of artery, diabetic ulcer, pressure ulcer, venous ulcer etc.Acute wounds can develop into chronic wounds.Acute wounds includes but not limited to the wound caused by such as thermal burn, wound, operation, large area skin carcinectomy, deep fungal and bacterium infection, vasculitis, scleroderma, pemphigus, toxic epidermal necrolysis etc.Referring to for example Buford, Wound Healing and Pressure Sores, Healing Well.com, publish October 24 calendar year 2001." normal wound " refers to experience the wound that normal wound healing is repaired.

" the growth-inhibiting amount " of polypeptide of the present invention, antibody, antagonist or composition refers to can be in vitro or suppress in vivo cell, especially tumour, for example amount of growth of cancer cells.Polypeptide of the present invention, antibody, antagonist or composition can be by rule of thumb for " the growth-inhibiting amount " that suppress neoplastic cell growth and are determined by currently known methods or the example that provides herein.

" the cytotoxicity amount " of polypeptide of the present invention, antibody, antagonist or composition refers to can be in vitro or cause in vivo cell, especially tumour, the amount that for example cancer cells destroys.Polypeptide of the present invention, antibody, antagonist or composition can be determined by rule of thumb and by means known in the art for " the cytotoxicity amount " that suppress the neoplastic cell growth.

" autoimmune disease " refer in this article to cause because of individual autologous tissue and for the disease of individual autologous tissue or illness or its, be divided into from (co-segregate) or performance or consequent illness.The example of autoimmune disease or illness includes but not limited to arthritis, and (rheumatoid arthritis is such as acute arthritis, chronic rheumatoid arthritis, urarthritis, acute gouty arthritis, chronic inflammatory arthritis, DA, infectional arthritis, lime (Lyme) arthritis, hypertrophic arthritis, psoriasis arthropathica, arthritis vertebralis and young hair style rheumatoid arthritis, osteoarthritis, chronic progressive external arthritis (arthritis chronica progrediente), arthritis deformans, chronic primary panarthritis, adjuvant arthritis, and ankylosing spondylitis), inflammatory hyper-proliferative dermatoses, psoriasis is such as plaque psoriasis, psoriasis guttata, pustular psoriasis and nail psoriasis, dermatitis comprises contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis and atopic dermatitis, the high IgM syndrome that x is chain, nettle rash such as chronic idiopathic urticaria comprises chronic autoimmune urticaria, polymyositis/dermatomyositis, JDMS, toxic epidermal necrolysis, chorionitis (comprising systemic scleroderma), sclerosis is such as systemic sclerosis, multiple sclerosis (MS) is such as spinal cord-eye (spino-optical) MS, primary carrying out property MS and disappearing property of recurrent (relapsingrcmitting) MS, the systemic sclerosis of carrying out property, atherosclerotic, artery sclerosis, disseminated sclerosis (sclerosis disseminata) and incoordination (ataxic) sclerosis, inflammatory bowel disease (IBD) (Crow engler (Crohn) disease for example, colitis such as ulcerative colitis (ulcerative colitis, colitis ulcerosa), microcosmic (microscopic) colitis, collagenous colitis, colitis polyposa, NEC and transmural colitis, with the LADA inflammatory bowel disease), PG, erythema nodosum, primary sclerotic cholangitis, episcleritis), Respiratory Distress Syndrome(RDS) comprises adult type or ARDS (ARDS), meningitis, uveal inflammation all or in part, iritis, choroiditis, LADA hematology illness, rheumatoid, sudden hearing loss, the disease of IgE mediation is such as allergic reaction and allergia and atopic rhinitis, encephalitis such as Lars write from memory Sen Shi (Rasmussen) encephalitis and edge system and/or BBE, uveitis is such as anterior uveitis, AAU, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis or LADA uveitis, the glomerulonephritis (GN) that has and do not have a nephrotic syndrome such as chronic or acute glomerulonephritis is such as primary GN, immune-mediated GN, film GN (membranous nephropathy), idiopathic film GN, film proliferative GN (MPGN) comprises I type and II type, with radical property GN, the allergia illness, allergic reaction, eczema comprises allergia or AE, asthma such as bronchial astehma (asthma bronchiale, bronchial asthma) and LADA asthma, relate to the illness that T cellular infiltration and chronic inflammatory are replied, chronic pulmonary inflammation disease, autoimmune myocarditis, leukocyte adhesion deficiency, systemic loupus erythematosus (SLE) (systemic lupuserythematosus, systemic lupus erythematodes) such as skin S LE, subacute cutaneous lupus erythema tosus, neonatal lupus syndrome (NLE), lupus erythematosus disseminatus, lupus (comprises lupus nephritis, lupus encephalitis, the paediatrics lupus, non-kidney lupus, discoid lupus, the lupus alopecia), children's hair style (I type) diabetes comprise paediatrics IDD (IDDM), the diabetes (type ii diabetes) of adulthood outbreak, autoimmune diabetes, idiopathic diabetes insipidus, the immune response relevant with the acute and delayed hypersensitivity of cell factor and T-cell mediated, tuberculosis, sarcoidosis, granulomatosis comprises lymphomatoid granulomatosis, Wei Genashi (Wegener) granulomatosis, agranulocytosis, vasculitides comprises that vasculitis (comprises trunk vasculitis (comprising polymyalgia rheumatica and giant cell (high iS-One (Takayasu)) arteritis), medium vessels vasculitis (comprising Chuan Qishi (Kawasaki) disease and PAN), microcosmic (microscopic) panarteritis, the CNS vasculitis, gangrenosum acne, skin or allergic angiitis, systemic necrotizing vasculitis, with ANCA related artery inflammation such as Qiu-Shi Er Shi (Churg-Strauss) vasculitis or syndrome (CSS)), temporal arteritis, alpastic anemia, LADA alpastic anemia, the positive anaemia of Claire (Coombs), Dai-Bu Er Shi (Diamond Blackfan) anaemia, hemolytic anemia or immune hemolytic anemia comprise autoimmune hemolytic anemia (AIHA), pernicious anaemia (pernicious anemia, anemia perniciosa), A Disenshi (Addison) disease, PRCA or aregeneratory (PRCA), Factor IX lacks, hemophilia A, the LADA neutrophil cell reduces disease, pancytopenia, leukopenia, the disease that relates to leukocyte infiltration, the CNS inflammatory conditions, multiple organ injury's syndrome is such as those septicemia, wound or hemorrhage secondary, the disease of antigen-antibody complex mediation, the anti-GBM disease, antiphospholipid antibody syndrome, allergic neuritis, Bei Qieteshi (Bechet or Behcet) disease, Ka Siermanshi (Castleman) syndrome, Gu Depasiqiushi (Goodpasture) syndrome, Lei Nuoshi (Reynaud) syndrome, Si Yegelunshi (Sjogren) syndrome, Er Shi (Stevens-Johnson) syndrome of Shi-Yue, pemphigoid is such as bullous pemphigoid and skin pemphigoid, pemphigus (comprises pemphigus vulgaris, pemphigus foliaceus, MMP type pemphigus and pemphigus erythematosus), autoimmune polyendocrinopathy, Lay Te Shi (Reiter) disease or syndrome, immune complex nephritis, antibody-mediated ephritis, chronic neuropathic is such as the neuropathy of IgM polyneuropathy or IgM mediation, thrombopenia (for example myocardial infarction patient occurs) comprises that thrombotic thrombocytopenic purpura (TTP) and autoimmunity or immune-mediated thrombopenia such as ITP (ITP) comprise chronic or acute ITP, the autoimmunity disease of testis and ovary comprises LADA orchitis and oaritis, primary hypothyroidism disease, hypoparathyroidism, autoimmune endocrinopathy comprises that thyroiditis is such as autoimmune thyroiditis, Hashimoto (Hashimoto) disease, chronic thyroiditis (Hashimoto (Hashimoto) thyroiditis) or subacute thyroiditis, AITD, the idiopathic hypothyroidism, Ge Leifusishi (Graves) disease, polyglandular syndrome such as autoimmune polyglandular syndrome (or polyadenous endocrine disease syndrome), paraneoplastic syndrome comprises neurology paraneoplastic syndrome such as Lambert-Eton (Lambert-Eaton) myasthenic syndrome or Eaton-Lambert (Lambert-Eaton) syndrome, stiff body or stiff man syndrome, encephalomyelitis such as allergic encephalitis (allergic encephalomyelitis, encephalomyelitisallergica) and EAE (EAE), myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonic syndrome (OMS), and esthesioneurosis, seat Han Shi (Sheehan) syndrome, oneself immunity hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, CAH or ACAH, LIP, bronchiolitis obliterans (Nonimplantation) is to NSIP, Ge-Ba Er Shi (Guillain-Barr é) syndrome, Bei Geershi (Berger) sick (IgA ephrosis), idiopathic IgA ephrosis, linear IgA skin disease, PBC, pneumonocirrhosis, auto immune enteropathy syndrome, chylous diarrhea, CD, sprue (gluten enteropathy), the intractable sprue, the idiopathic sprue, cryoglobulinemia, ALS (ALS) (Lu Gelikeshi (Lou Gehrig) disease), coronary artery disease, Autoimmune Inner Ear Disease (AIED) or LADA anaudia, opsoclonus myoclonic syndrome (OMS), polychondritis is such as intractable or relapsing polychondritis, pulmonary alveolar proteinosis, amyloidosis, sclerotitis, non-carcinous lymphocytosis, primary lymphocytosis comprises that monoclonal B cell lymphocyte increases (for example undetermined MG of benign monoclonal gammopathy and character (MGUS)), peripheral nerve disease, paraneoplastic syndrome, the passage disease is such as epilepsy, antimigraine, arrhythmia cordis, disorder of muscle, become deaf, blind, the passage disease of periodic paralysis and CNS, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, the uvea retinitis, choroidoretinitis, LADA hepatology illness, fibromyalgia, multiple endocrinasthenia, Shi Miteshi (Schmidt) syndrome, paranephritis, lipogastry, alzheimer's disease, demyelinating disease is such as the LADA demyelinating disease, diabetic nephropathy, De Leisileshi (Dressler) syndrome, alopecia areata, CREST syndrome (calcinosis, Lei Nuoshi (Raynaud) phenomenon, esophageal dysmotility, sclerodactyly and capillarectasia), the masculinity and femininity LADA is infertile, MCTD, just add Si Shi (Chagas) disease, rheumatic fever, habitual abortion, farmer lung, erythema multiforme, postcardiotomy syndrome, Ke Xing Shi (Cushing) syndrome, the bird breeders' lung, allergic granulomatous angiitis, optimum lymphocytic vasculitis, A Erboteshi (Alport) syndrome, pulmonary alveolitis is such as allergic pulmonary alveolitis and FA, interstitial lung disease, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis (kypanosomiasis), snail fever, roundworm disease, aspergillosis, the Sampter Cotard, Kapp Lan Shi (Caplan) syndrome, step on leather, endocarditis, endomyocardial fibrosis, diffuse pulmonary interstitial fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, entophthamia, erythema elevatum diutinum (erythema elevatum et diutinum), EBF, eosinocyte fascitis (faciitis), Shu Er Man (Shulman) syndrome, Fil Ti Shi (Felty) syndrome, flariasis, cyclitis is such as chronic cyclitis, the heterochronia cyclitis, iridocyclitis or FuchShi cyclitis, Heng Nuo-Xu Lan Er Shi (Henoch-Schonlein) purpura, human immunodeficiency virus (HIV) infects, echovirus infects, cardiomyopathy, Alzheimers (Alzheimer) disease, parvovirus infections, rubella virus infection, syndrome after vaccination, congenital rubella infects, Epstein-Ba Er (Epstein-Barr) virus infections, mumps, Ai Wensi (Evans) syndrome, the exhaustion of LADA sexual gland, Xi Denghamushi (Sydenham) chorea, ephritis after streptococcus, Buerger's disease (thromboangitisubiterans), thyrotoxicosis, tabetic crisis, choroiditis, the megaloblastic polymyalgia, endocrine ophthalmopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivities, the idiopathic nephritic syndrome, MCN, benign familial and ischemia reperfusion injury, retina autoimmunity, arthritis, bronchitis, chronic obstructive airway disease, silicosis, aphtha, aphthous stomatitis, the arteriosclerotic illness, without spermatogenesis (aspermiogenese), autoimmune hemolytic anemia, uncle Ke Shi (Boeck) disease, cryoglobulinemia, dupp Yi Telunshi (Dupuytren) contracture, Phacoanaphylaxis entophthamia (endophthalmia phacoanaphylactica), allergia enteritis (enteritis allergica), leprosy joint erythema nodosum, Idiopathic facial palsy, chronic tired syndrome, rheumatic fever (febris rheumatica), Ha-inner Er Shi (Hamman-Rich) disease, sensory neural hearing loss, paroxysmal hemoglobinuria (haemoglobinuria paroxysmatica), hypogonadism, regional enteritis (ileitisregionalis), leukopenia, infectious mononucleosis, traversing property (traverse) myelitis, primary idiopathic myxoedema, ephrosis, sympathetic ophthalmia (ophthalmia symphatica), granulomatous orchitis (orchitis granulomatosa), pancreatitis, acute polyradiculitis, PG, Kui Erwanshi (Quervain) thyroiditis, acquired splenatrophy, sterility due to anti-spermatozoon antibody, non-malignant thymoma, leucoderma, the viral relevant disease of SCID and Epstein-Ba Er (Epstein-Barr), acquired immunodeficiency syndrome (AIDS), parasitic disease is such as Leishmania, TSS, food poisoning, the illness that relates to the T cellular infiltration, leukocyte adhesion deficiency, the immune response relevant with the acute and delayed hypersensitivity (DH) of cell factor and T-cell mediated, the disease that relates to leukocyte infiltration, multiple organ injury's syndrome, the disease of antigen-antibody complex mediation, the anti-GBM disease, allergic neuritis, autoimmune polyendocrinopathy, oaritis, the primary myxoedema, the LADA atrophic gastritis, sympathetic ophthalmia, rheumatism, MCTD, nephrotic syndrome, inflammation of pancreatic islet, many endocrinasthenias, peripheral nerve disease, autoimmune polyglandular syndrome I type, the Idiopathic hypoparathyroidism (AOIH) of adulthood outbreak, whole alopecia, dilated cardiomyopathy, epidermolysis bullosa acquisita (epidermolisis bullosa acquisita, EBA), hematochromatosis, myocarditis, nephrotic syndrome, primary sclerotic cholangitis, suppurative or apyetous nasosinusitis, acute or chronic nasosinusitis, eso-ethmoiditis, frontal sinusitis, maxillary sinusitis or sphenoiditis, the eosinocyte associated conditions is such as eosinophilia disease, the ensinophilosis infiltration, eosinophilia-muscle pain syndrome, Lv Fuleshi (Loffler) syndrome, chronic eosinocyte pneumonia, torrid zone ensinophilosis, BPA, aspergilloma, or contain eosinophilic granuloma, allergic reaction, seronegativity arthritis vertebralis disease, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, the chronic mucocutaneous candidiasis, Bu Ludunshi (Bruton) syndrome, infancy transient hypogammaglobulinemia, neat (Wiskott-Aldrich) syndrome in prestige Scott-Ao Er Delhi, incoordination capillarectasia, the autoimmune conditions relevant with the following: collagen disease, rheumatism, neurological disease, the ischemia-reperfusion disorder, blood pressure is replied reduction (reduction in blood pressure response), dysfunction of blood vessel, capillarectasia (antgiectasis), tissue damage, cardiovascular ischemic, hyperalgia, cerebral ischemia and the disease of following vascularization, allergia supersensitivity illness, the glomerulonephritis disease, reperfusion injury, the reperfusion injury of cardiac muscle or other tissue, skin disease with acute inflammation composition, acute purulent meningitis or other central nervous system inflammatory conditions, the granulocyte Transfusion related syndromes, it is poisoning that cell factor is brought out, the acute severe inflammation, the chronic and refractory inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic keratopathy main artery illness, hyperplasia in artery, peptic ulcer, cardiovalvulitis, and endometriosis.

Cancer therapy can be by shrinking such as but not limited to tumor regression, gross tumor volume or size, assessing apart from the time of progress, survive time length, progresson free survival, Whole Response rate, duration of response, quality of life, protein expression and/or activity.Therefore need not to be neoplastic cell itself because of antiangiogenic agent target tumor blood vessel structure described herein, so they have represented the anticarcinogen of a class uniqueness, and can require measurement and the definition of unique clinical response to medicine.For example, being greater than in two-dimension analysis that 50% tumour shrinks is that the standard of declaration response is held back.Yet alpha 5 beta 1 antagonists of the present invention and VEGF antagonist can cause the contraction that the inhibition of transitivity diffusion be there is no to primary tumor, or can only bring into play the inhibition tumor effect.Thereby, can adopt the method for measuring therapeutic efficiency, comprise blood plasma or the urine mark of for example measuring the blood vessel generation and respond by the radiology imaging measurement.

Depend on the factor relevant with dosed administration that indication to be treated is familiar with this area skilled practitioners, with the dosage of effectively treating this indication simultaneous minimization Side effect, use antibody of the present invention.In order to treat cancer, autoimmune disease or immunodeficient disease, the treatment significant quantity can arrive in the scope of 2.5g/m2 in for example 50mg/ agent.In one embodiment, the dosage of using is that about 250mg/m2 is to about 400mg/m2 or 500mg/m2.In another embodiment, dosage is about 250-375mg/m2.In another embodiment, dosage range is 275-375mg/m2.

The treatment of age related macular degeneration (AMD) can be through but not limited to further the reduction of visual loss ratio/speed or the prevention of further visual loss are assessed.For the AMD therapy, effect can be by for example following one or more the measurement in body: assessment preferably correct defects of vision (BCVA) from baseline the Change in Mean to the expectation time; Being evaluated at the expected time compares experimenter's ratio that vision loss is less than 15 letters with baseline; Being evaluated at the expected time compares experimenter's ratio that eyesight to surpass or equal 15 letters with baseline; Be evaluated at expeced time Snellen eyesight and be equivalent to 20/2000 or worse experimenter's ratio; Assessment NEI sight function questionnaire (Visual Functioning Questionnaire); The size of assessment CNV expeced time and the amount of CNV seepage, assess by FA; Deng.

Term " detection " intention comprise measure material existence whether or quantitative to amount of substance.This term so refers to material of the present invention, composition and method for quantitative and qualitative test.Generally speaking, for detection of concrete technology for enforcement of the present invention, be not vital.

For example, according to " detection " of the present invention, can comprise: whether the existence of observation α 5 gene products, mRNA molecule or α 5 polypeptide; α 5 polypeptide levels or be bonded to the variation of the amount of target thing; The variation of the biological function of α 5 polypeptide/activity.In some embodiments, " detection " can comprise wild-type α 5 levels (for example mRNA or polypeptide level) that detect.Detection can comprise to compared with the control between any numerical value between 10% and 90% or between 30% and 60% any numerical value or surpass 100% variation (raise or reduce) quantitatively.Detection can comprise to (containing endpoints thereof) any numerical value between 2 times and 10 times or larger (for example 100 times) variation quantitatively.

Term " marker " refers to detectable compounds or the composition with the direct or indirect coupling of antibody for this paper the time.Marker self can be by self for example, with regard to detectable (radioisotopic tracer or fluorescent marker), or in the situation of enzyme labelling thing, but the chemically changed of the detectable substrate compounds of catalysis or composition.

New anti-alpha 5 beta 1 antibody

Providing herein can be in conjunction with the new antibodies of people α 5 β 1 competitive inhibition anti-alpha 5 beta 1 antibodies people α 5 β 1.According to an embodiment, described anti-alpha 5 beta 1 antibody is to be generated by the hybridoma that is selected from lower group: on March 7th, 2006 at ATCC as the hybridoma of α 5/ β 1 7H5.4.2.8 (ATCC No.PTA-7421) preservation with as the hybridoma of α 5/ β 1 7H12.5.1.4 (ATCC No.PTA-7420) preservation.According to another embodiment, described antibody is to be generated by the hybridoma that is selected from lower group: on March 7th, 2006 at ATCC as the hybridoma of α 5/ β 1 7H5.4.2.8 (ATCC No.PTA-7421) preservation with as the hybridoma of α 5/ β 17H12.5.1.4 (ATCC No.PTA-7420) preservation.According to another embodiment, heavy chain variable domain (VH) and light chain variable territory (VL) sequence that described antibody comprises the antibody generated as the hybridoma of α 5/ β 1 7H5.4.2.8 (ATCC No.PTA-7421) preservation at ATCC by March 7th, 2006.In another embodiment, described antibody comprises the antibody generated as the hybridoma of α 5/ β 1 7H12.5.1.4 (ATCC No.PTA-7420) preservation at ATCC by March 7th, 2006 heavy chain variable domain (VH) and light chain variable territory (VL) sequence.The people's antibody or the chimeric antibody form that also contain the antibody of institute's preservation hybridoma.

According to an embodiment, described antibody capable with the Kd between 500nM and 1pM in conjunction with people α 5 β 1.According to another embodiment, described antibody can not be in conjunction with α V β 3 or α V β 5 or α V β 1.According to another embodiment, described antibody comprises human IgG, for example the Fc sequence of human IgG1 or human IgG 4.In another embodiment, the Fc sequence has changed or has had alternate manner to change, and makes its lack cytotoxicity (ADCC) effector functions of antibody dependent cellular, this usually with they to Fc acceptor (FcR) in conjunction with relevant.Fc sequence variation or the sudden change that can change effector functions have many examples.For example, WO00/42072 (Presta) and Shields et al.J.Biol.Chem.9 (2): 6591-6604 (2001) have put down in writing the antibody variants with FcR combination raising or that reduce.The content of these publications clearly is collected herein by reference.Described antibody can be Fab, Fab ', F (ab) ' ₂, scFv (scFv), Fv fragment; The form of double antibody and linear antibody.Equally, described antibody can be can be in conjunction with the multi-specificity antibody of α 5 β 1, and is alpha 5 beta 1 antagonists, but can also and suppress its function (for example VEGF) in conjunction with one or more other target things.Described antibody can be coupled to therapeutical agent (for example cytotoxic agent, radio isotope and chemotherapeutics) or marker, the latter for detection of α 5 β 1 in patient's sample or by imaging for example, for detection of 5 β 1 of α in body (radio isotope, fluorescence dye and enzyme).

Also contain the nucleic acid molecule of coding anti-alpha 5 beta 1 antibody, the expression vector of nucleic acid molecule that comprises two kinds of variable domains of coding one or both of and the cell that comprises described nucleic acid molecule.These antibody can be used for therapy described herein and for example, for detection of (FACS, immunohistochemical methods (IHC), ELISA assay method) in patient's sample or α 5 β 1 albumen in the patient.

New composition

Provide the blood vessel of the experimenter for suppressing to suffer from disease to occur herein and/or the novel composition of vascular permeability, said composition comprises VEGF antagonist and alpha 5 beta 1 antagonists.Described VEGF antagonist and described alpha 5 beta 1 antagonists can or be used in turn in the treatment circulation simultaneously.This type of conjoint therapy is useful for the treatment disease, comprises the disease that those have abnormal vascular generation and/or vascular permeability and will benefit from anti-angiogenic generation therapy.This type of disease includes but not limited to cancer, illness in eye and autoimmune disease.Perhaps, the experimenter can use described alpha 5 beta 1 antagonists subsequently with described VEGF antagonist for treating, for example uses the VEGF antagonist for treating, until the experimenter does not respond the VEGF antagonist for treating, then the experimenter treats with alpha 5 beta 1 antagonists.According to an embodiment, the experimenter is standby VEGF antagonist for treating when the cancer right and wrong are invasive, and in cancer standby alpha 5 beta 1 antagonists treatment while being invasive.Some with without the patient or contrast the patient who compares α 5 β 1 levels that natural experience raises or response VEGF antagonist therapy and experience α 5 β 1 levels of rising and especially this conjoint therapy is had to response.Contain the composition that further comprises therapeutical agent (for example antineoplastic agent, chemotherapeutics, growth inhibitor and cytotoxic agent).For example, there are stand-by chemotherapy (for example irinotecan) and the patient of alpha 5 beta 1 antagonists treatment or the patient who had treated with chemotherapy and alpha 5 beta 1 antagonists can benefit from VEGF antagonist therapy.Perhaps, the patient who has crossed with chemotherapy and VEGF antagonist for treating can benefit from the alpha 5 beta 1 antagonists therapy.In a preferred embodiment, described VEGF antibody is

antibody.In another preferred embodiment, described anti-alpha 5 beta 1 antibody is anti-alpha 5 beta 1 antibody described herein.Contain the test kit that comprises VEGF antagonist, alpha 5 beta 1 antagonists and optional chemotherapeutics.

Medicinal proportional preparation

The treatment of the antibody used according to the present invention is the antibody by will have expectation purity and optional pharmaceutically acceptable carrier, vehicle or stablizer (Remington ' s PharmaceuticalSciences with preparaton, the 16th edition, Osol, A. compile (1980)) mix, with the form preparation of freeze-dried formulation or the aqueous solution, store altogether.Acceptable carrier, vehicle or stablizer are nontoxic at adopted dosage and concentration to the recipient, comprise buffer reagent, such as phosphoric acid salt, Citrate trianion and other organic acid; Antioxidant, comprise xitix and methionine(Met); Sanitas is (such as octadecyl dimethyl benzyl ammonium chloride; Chlorination hexane diamine; Benzalkonium chloride, benzethonium chloride; Phenol, butanols or phenylcarbinol; Alkyl paraben, such as methyl esters or the propyl ester of P-hydroxybenzoic acid; Pyrocatechol; Resorcinol; Hexalin; The 3-amylalcohol; And meta-cresol); Lower molecular weight (being less than approximately 10 residues) polypeptide; Protein, such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer, such as polyvinylpyrrolidone; Amino acid, such as glycine, glutamine, l-asparagine, Histidine, arginine or Methionin; Monose, disaccharides and other carbohydrate, comprise glucose, seminose or dextrin; Sequestrant, such as EDTA; Carbohydrate, such as sucrose, N.F,USP MANNITOL, trehalose or sorbyl alcohol; The salify gegenion, such as sodium; Metal composite (for example Zn-protein complex); And/or nonionogenic tenside, such as TWEEN ^tM, PLURONICS ^tMor polyoxyethylene glycol (PEG).Exemplary antibody formulations is recorded in WO 98/56418, at this, clearly takes in as a reference.The freeze-dried formulation that is suitable for subcutaneous administration is recorded in WO 97/04801.This type of freeze-dried formulation can be rebuild to increased protein concentration with suitable thinner, but the preparaton subcutaneous administration of reconstruction is in Mammals to be treated herein.

Preparaton herein also can contain and surpass the necessary active compound of a kind of treat concrete indication, preferably those active complementations and there is no each other the compound of disadvantageous effect.For example, may wish further to provide cytotoxic agent, chemotherapeutics, cytokine or immunosuppressor (for example acting on the immunosuppressor of T cell, such as ciclosporin or in conjunction with the antibody of T cell, for example, in conjunction with the antibody of LFA-1).The significant quantity of this type of other medicament depends on type, and the other factors discussed above of amount, disease or the illness of the antibody existed in preparaton or treatment.These normally with identical dosage described herein, with the path of using described herein, use, or about 1-99% of the dosage that adopted so far.

Activeconstituents also can wrap and for example for example be stated from, by condensation technique or the microcapsule that prepare by interfacial polymerization (being respectively Walocel MT 20.000PV or gelatin microcapsule and poly-(methyl methacrylate) microcapsule), for example, in gluey drug delivery system (liposome, white protein microsphere, microemulsion, nano particle and Nano capsule), or in macro emulsion.This type of technology is disclosed in for example Remington ' sPharmaceutical Sciences, and the 16th edition, Osol, A. compiles (1980).

Can prepare extended release preparation.The suitable example of extended release preparation comprises the solid hydrophobic polymkeric substance semipermeability matrix that contains antagonist, and this matrix is the form of standardized product, for example film or microcapsule.The example of sustained release matrix comprises polyester, hydrogel (for example poly-(2-hydroxyethyl-methacrylic ester) or poly-(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), the multipolymer of Pidolidone and Pidolidone ethyl ester, nondegradable ethane-acetic acid ethyenyl, degradable lactic acid-ethanol copolymer are such as LUPRON DEPOT ^tM(the Injectable microspheres body formed by lactic acid-ethanol copolymer and leuprorelin acetate) and poly--D-(-)-3-hydroxybutyrate.

The preparaton that is used for using in body must be aseptic.This can be easy to by realizing with aseptic membrane filtration.

Goods and test kit

Another embodiment of the invention is the goods that comprise the material that can be used for treating tumour, illness in eye or autoimmune disease and related disorders.Described goods can comprise on container and container or the label relevant to container or package insert.Suitable container comprises such as medicine bottle, pencil, syringe etc.Described container can be made with multiple material, such as glass or plastics.Generally speaking, described container is equipped with the composition of the described illness of effective treatment, can have aseptic access port (for example described container can be the medicine bottle of intravenous solution bag or the stopper that can pierce through with hypodermic needle).At least one active agents in described composition is VEGF antagonist of the present invention or alpha 5 beta 1 antagonists or VEGFR agonist or α 5 β 1 agonists.Described label or package insert indicate said composition and are used for the treatment of described concrete illness.Described label or package insert further comprise about the patient being used to the specification sheets of described antibody compositions.Also contain the goods and the test kit that comprise conjoint therapy described herein.

Package insert refers to be usually included in the specification sheets for the treatment of in pack with product ommercialization, the information that it comprises the indication of product use for relevant this type for the treatment of, usage, dosage, uses, avoids and/or warn.In one embodiment, package insert indicates said composition and is used for the treatment of non_hodgkin lymphoma.

In addition, described goods can further comprise second container, pharmacy wherein is housed and can accepts damping fluid, such as injection bacteriostatic water (BWFI), phosphate buffered saline (PBS), woods Ge Shi (Ringer) solution and dextrose solution.It can further comprise from other material of business and user's position needs, comprise other damping fluid, thinner, filter, pin and syringe.

The test kit that can be used for various purposes also is provided, for example for separating of or detect α 5 β 1 and/or the VEGF in the patient, optionally combine described goods.In order to separate and purification of alpha 5 β 1, described test kit can comprise for example, anti-alpha 5 beta 1 antibody with pearl (sepharose pearl) coupling.Can provide and comprise for α 5 β 1 and/or VEGF vitro detection and quantitative antibody, for example carry out in ELISA or Western trace.The same with goods, described test kit comprises on container and container or the label relevant to container or package insert.For example, described container is equipped with the composition that comprises at least one anti-alpha 5 beta 1 antibody of the present invention.Can comprise for example other container of thinner and damping fluid, control antibodies is housed.Described label or package insert can provide the description of said composition and be intended to specification sheets external or that diagnosis is used.

antibody falls in single gram

Monoclonal antibody can be used for example hybridoma method to prepare, such as those by Kohler andMilstein, Nature, 256:495 (1975) record, perhaps can pass through recombinant DNA method (U.S. Patent No. 4,816,567) prepare, or can generate by the described method of this paper embodiment part.In hybridoma method, usually with immunizing agent immune mouse, hamster or other suitable host animal, to cause to generate, maybe can generate the lymphocyte of following antibody, described antibody is by the described immunizing agent of specific binding.Perhaps, immunological lymphocyte in vitro.

Immunizing agent generally includes polypeptide or the fusion rotein of proteins of interest matter or the composition that comprises this protein.Generally speaking, if the cell of wanting the people to originate from is used peripheral blood lymphocyte (PBL); Perhaps, if want the non-human mammal source, use splenocyte or lymph-node cell.Then, use suitable fusogen such as polyoxyethylene glycol that lymphocyte and immortalized cell line are merged, to form hybridoma (Goding, Monoclonal Antibodies:Principles and Practice, New Yourk, Academic Press, 1986, pp.59-103).Immortalized cell line is the myeloma cell of the mammalian cell, particularly rodents through transforming, ox and people's origin normally.Usually, adopt rat or mouse myeloma cell line.Hybridoma can be cultivated in suitable medium, and described medium optimization contains immortalized cells growth that inhibition do not merge or one or more materials of survival.For example, if parental cell lacks enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the substratum for hybridoma typically will contain xanthoglobulin, aminopterin-induced syndrome and thymidine (HAT substratum), and these materials stop the growth of HGPRT deficient cells.

Preferred immortalized cell line is that those efficiently merge, support high level expression antibody that selected antibody-producting cell is stable and to the substratum sensitivity such as the HAT substratum.Preferred immortalized cell line is rat bone marrow tumour system, it can be from for example Sol gram institute cell distribution center (Salk InstituteCell Distribution Center, San Diego, California, USA) and American type culture collection (American Type Culture Collection, Manassas, Virginia, USA) obtain.Human myeloma and mouse-people's allos myeloma cell line has also been put down in writing for generating human monoclonal antibodies (Kozbor, J.Immunol.133:3001 (1984); Brodeur et al., Monoclonal Antibody ProductionTechniques and Applications, Marcel Dekker, Inc., New York, 1987, pp.51-63).

Then the nutrient solution that can cultivate just therein hybridoma is measured the existence for the monoclonal antibody of polypeptide.Can, by immunoprecipitation or by external binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), measure the binding specificity of the monoclonal antibody generated by hybridoma.This type of technology and assay method are known in the art.The binding affinity of monoclonal antibody can be by for example Munson and Pollard, and the Scatchard of Anal.Biochem.107:220 (1980) analyzes to measure.

After identifying the hybridoma of wanting, this clone can carry out subclone and be cultivated (Goding sees above) by standard method by the limiting dilution rules.The substratum that is suitable for this purpose comprises for example DulbeccoShi improvement EagleShi substratum and RPMI-1640 substratum.Perhaps, hybridoma can carry out culturing in vivo as ascites in Mammals.

Can pass through routine immunization sphaeroprotein purifying rules, such as for example albumin A-Sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography, from the monoclonal antibody of nutrient solution or the secretion of ascites isolated or purified subclone.

Can also prepare by recombinant DNA method by monoclonal antibody, such as U.S. Patent No. 4,816, put down in writing in 567.The DNA of code book invention monoclonal antibody can be easy to use conventional rules to separate and order-checking (for example oligonucleotide probe of the gene by using can specific binding encode murine antibody heavy chain and light chain).Using the preferred source of hybridoma of the present invention as this type of DNA.Once separate, DNA can be placed in to expression vector, then this expression vector is transfected into and does not originally generate in the host cell of immunoglobulin (Ig) protein, such as ape and monkey COS cell, Chinese hamster ovary (CHO) cell or myeloma cell, to obtain the synthetic of monoclonal antibody in recombinant host cell.All right modifying DNA is for example encoding sequence replacement homology mouse sequence (U.S. Patent No. 4,816,567 of employment heavy chain and light chain constant domain by substituting; Morrison et al., see above), or by encoding sequence all or in part and the immunoglobulin coding sequence of NIg polypeptide is covalently bound.Can substitute with this type of NIg polypeptide the constant domain of antibody of the present invention, or can substitute the variable domain of an antigen binding site of antibody of the present invention with them, to create chimeric bivalent antibody.

Described antibody can be univalent antibody.Method for the preparation of univalent antibody is known in the art.For example, a kind of method involves recombinant expressed light chain immunoglobulin and modified heavy chain.Described heavy chain generally in the Fc district any site brachymemma crosslinked to prevent heavy chain.Perhaps, the cysteine residues of being correlated with substitutes with another kind of amino-acid residue or deletes crosslinked to prevent.

In vitro method also is applicable to prepare univalent antibody.Digestion antibody is to generate its fragment, and particularly the Fab fragment, can be used but not limited to technology known in the art and realize.

people's antibody and humanized antibody

Antibody can be humanized antibody or people's antibody.The humanization form of inhuman (for example mouse) antibody refers to that common bottom line comprises gomphosis immunoglobulin, immunoglobulin chain or its fragment derived from the sequence of non-human immunoglobulin (such as Fv, Fab, Fab ', F (ab ') ₂or other antigen zygote sequence of antibody).Humanized antibody comprises the antibody that the CDR residue in human normal immunoglobulin (receptor antibody) is replaced with the CDR residue of inhuman species (donor antibody) such as mouse, rat or rabbit with expectation specificity, avidity and ability.In some situation, the Fv framework residue of human normal immunoglobulin is replaced with corresponding inhuman residue.Humanized antibody also can be included in receptor antibody and input CDR or Frame sequence does not all have the residue of finding.Usually, humanized antibody can comprise at least one, common two whole following variable domains basically, wherein all or basically all CDR corresponding to the CDR of non-human immunoglobulin, and all or basically all FR are FR of human normal immunoglobulin consensus sequence.Humanized antibody preferably also will comprise at least part of constant region for immunoglobulin (Fc), normally the constant region of human normal immunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); Presta, Curr.Op.Struct.Biol.2:593-596 (1992)).

For humanization non-human antibody's certain methods this area and hereinafter embodiment description is arranged.Usually, one or more amino-acid residues from inhuman source have been introduced in humanized antibody.These inhuman amino-acid residues are often referred to as " input " residue, and they take from " input " variable domain usually.According to an embodiment, humanization can basically be followed Winter and colleague's thereof method and carry out (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), use the corresponding sequence of rodents CDR or CDR sequence replacing people antibody.Therefore, this type of " humanization " antibody is such antibody (U.S. Patent No. 4,816,567), wherein basically is less than whole people's variable domain and uses the corresponding sequence from inhuman species to substitute.In practice, normally some of them CDR residue and possible some FR residues people antibody alternative from the residue in similar site in rodents antibody of humanized antibody.

As humanized alternative method, can generate people's antibody.For example, likely generate such transgenic animal (for example mouse) now, they can generate the complete complete or collected works of people's antibody in the situation that lack the endogenous immunoglobulin generation after immunity.For example, put down in writing isozygotying of heavy chain of antibody joining region (JH) gene in chimeric and germ line mutation mouse and deleted the inhibition fully that causes endogenous antibody to generate.A large amount of people's germline immunoglobulin genes are transferred in this type of germ line mutation mouse and will cause generating people's antibody after antigen is attacked.Referring to for example Jakobovits et al., Proc.Natl.Acad.Sci.USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year inImmuno.7:33 (1993); U.S. Patent No. 5,545,806,5,569,825,5,591,669 (all belonging to GenPharm); 5,545,807; WO 97/17852.Perhaps, can generate as follows people's antibody, be about to human immunoglobulin gene's seat and import transgenic animal, for example the endogenous immunoglobulin gene mouse of deactivation partially or completely.When attacking, the generation of observing people's antibody in all respects in the people, see closely similar, comprise gene rearrangement, assembling and antibody complete or collected works.This method is recorded in for example U.S. Patent No. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; With 5,661,016, and following scientific publications: Marks et al., Bio/Technology, 10:779-783 (1992); Lonberg etal., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, NatureBiotechnology, 14:826 (1996); Lonberg and Huszar, Intern.Rev.Immunol., 13:65-93 (1995).

Perhaps, display technique of bacteriophage (McCafferty et al., Nature 348:552-553 (1990)) is used in external from from immunoglobulin variable territory (V) the gene complete or collected works of epidemic disease donor rather, generating people's antibody and antibody fragment.According to an embodiment of this technology, the antibody variable domains sequence is cloned in the main or less important coat protein gene of filobactivirus such as M13 or fd in the mode that meets reading frame, and is shown as the functional antibodies fragment on the phage particle surface.Phage display can carry out in a variety of forms, for example hereinafter the embodiment part is described, or summarizes referring to for example Johnson Kevin S.andChiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V constant gene segment C can be used for phage display.Clackson et al., Nature 352:624-628 (1991) isolates a large amount of different Kang azolactone antibody from the small-sized V gene random combine library derived from through the immune mouse spleen.Can basically follow Marks et al., J.Mol.Biol.222:581-597 (1991) or Griffith et al., the EMBO technology that J.12:725-734 (1993) are put down in writing, build V gene complete or collected works and Separated pin to the antibody of synantigen (comprising autoantigen) not in a large number by not immune people's donor.Also can be referring to U.S. Patent No. 5,565,332 and 5,573,905.

As mentioned above, also can be by the Activation In Vitro B cell antibody (referring to U.S. Patent No. 5,567,610 and 5,229,275) of being grown up next life.

People's antibody can also be produced by multiple technologies known in the art, comprises phage display library (Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991); Marks et al., J.Mol.Biol., 222:581 (1991)).The people's such as the people such as Cole and Boerner technology also can be used for preparing human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985) and Boerner et al., J.Immunol., 147 (1): 86-95 (1991)).

multi-specificity antibody

Multi-specificity antibody refer to two or more not synantigen there is the monoclonal antibody of binding specificity, preferably people's antibody or humanized antibody (for example, bi-specific antibody has binding specificity at least two kinds of antigens).For example, one of binding specificity can be for alpha 5 beta 1 antibodies, and another of binding specificity can be for any other antigen.According to a preferred embodiment, another antigen is cell surface protein or acceptor or receptor subunit.For example, described cell surface protein can be NK cell (NK) cell receptor.So, according to an embodiment, bi-specific antibody of the present invention can be in conjunction with α 5 β 1 with in conjunction with VEGF.

The existing description of example for the method that builds bi-specific antibody.Traditional, the coexpression of the recombinant production of bi-specific antibody based on two pairs of heavy chain immunoglobulin/light chains, wherein two kinds of heavy chains have different specificity (Millstein and Cuello, Nature 305:537-539 (1983)).Due to the random assignment of heavy chain immunoglobulin and light chain, these hybridomas (four source hybridomas (quadroma)) generate the potential mixture of 10 kinds of different antibodies molecules, wherein only have a kind of correct dual specific structure that has.Usually carry out the purifying of correct molecule by the affinity chromatography step.Similarly rules are disclosed in WO 93/08829 and the Traunecker et al. announced on May 13rd, 1993, and EMBO is (1991) J.10:3655-3659.

Antibody variable domains and immunoglobulin (Ig) constant domain sequence with expectation binding specificity (antibody-antigen binding site) can be merged.Preferably, with the heavy chain immunoglobulin constant domain that comprises at least part of hinge, CH2He CH3 district, merged.Preferably, exist and comprise first CH (CH1) of light chain in conjunction with necessary site at least one fusions.To encode the heavy chain immunoglobulin fusions and, when needed, the DNA of light chain immunoglobulin inserts expression vector separately, and cotransfection is in the appropriate host organism.About the further details that generate bi-specific antibody referring to for example Suresh et al., Methods in Enzymology 121:210 (1986).

Also put down in writing from the recombinant cell culture thing and directly generated and the multiple technologies of separating bispecific antibody fragment.For example, used leucine zipper to generate bi-specific antibody.Kostelny et al.，J.hmmunol.148(5)：1547-1553(1992)。To by gene fusion, with the Fab ' part of two kinds of different antibodies, be connected from the leucine zipper peptide of Fos and Jun albumen.The antibody homodimer reduces to form monomer at hinge area, then again oxidation to form the antibody heterodimer.This method also can be used for generating the antibody homodimer.Hollinger et al., " double antibody " technology of Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993) record provides the replacement mechanism that builds bispecific antibody fragment.This fragment comprises the VH connected by joint and VL, between too short two structural domains that make on the same chain of described joint, can not match.Therefore, force VH and the complementary VL on VL structural domain and another fragment and a pairing of VH structural domain on fragment, form thus two antigen binding sites.Also reported by using scFv (sFv) dimer to build the another kind strategy of bispecific antibody fragment.Referring to Gruber et al., J.Immunol.152:5368 (1994).

Imagined and there are two antibody of tiring of surpassing.For example, can prepare three-specific antibody.Tutt et al.，J.Immunol.147：60(1991)。

allos coupling antibody

Allos coupling antibody consists of two kinds of covalently bound antibody.This antibody-like for example is proposed to be used in the undesired cell of immune system cell target (U.S. Patent No. 4,676,980) and is used for the treatment of HIV and infects that (WO 91/00360; WO 92/200373; EP 03089).Imagined the currently known methods Dispersal risk that can use in vitro the synthetic protein chemistry, comprised that those relate to the method for linking agent.For example, can build immunotoxin with the disulfide exchange reaction or by forming thioether bond.The example that is suitable for the reagent of this purpose comprises imino-mercaptan ester/salt (iminothiolate) and 4-sulfydryl butyryl imido acid methyl esters (methyl-4-mercaptobutyrimidate) and for example disclosed in U.S. Patent No. 4,676,980.

effector functions is engineered

May wish to modify antibody of the present invention aspect effector functions, thereby strengthen for example effect of antibody in the treatment cancer.For example, Ke Xiang Fc introduces cysteine residues in district, thereby make in the Gai district, forms interchain disulfide bond.The homodimer antibody so generated can have cell killing and the antibody dependent cellular cytotoxicity (ADCC) of the complement-mediated of the internalization ability of improvement and/or raising.Referring to Caron etal., J.Exp.Med.176:1191-1195 (1992) and Shopes, J.Immunol.148:2918-2922 (1992).Homodimer antibody with anti-tumor activity of enhancing also can be used the al. as Wolff et, prepared by the isodigeranyl functional cross-link agent of describing in Cancer Research 53:2560-2565 (1993).Perhaps, antibody can be transformed into has dual Fc district, and the complement that can have thus enhancing dissolves and the ADCC ability.Referring to Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

Can carry out the sudden change in the Fc region sequence or change for example improve FcR, in conjunction with (FcyR, FcRn).According to an embodiment, antibody of the present invention has the effector functions of group under being selected from of at least one change: ADCC, CDC and improved FcRn combination, with natural IgG or parental antibody, compare.The example of the specific sudden change that several are useful is recorded in for example Shields, RL et al. (2001) JBC 276 (6): 6591-6604; Presta, L.G., (2002) Biochemical Society Transactions 30 (4): 487-490; WO publication WO00/42072.

According to an embodiment, the Fc receptor mutation is to be selected from the substituting of at least one position of lower group: 238 of Fc district, 239, 246, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439, wherein the residue numbering in Fc district is according to the EU numbering system.

immune conjugate

The present invention also has the immune conjugate of the antibody of cytotoxic agent about comprising coupling, described cytotoxic agent such as chemotherapeutics, toxin (as enzyme activity toxin or its fragment of bacterium, fungi, plant or animal origin) or radio isotope (radiating conjugate).

The chemotherapeutics that can be used for generating this type of immune conjugate has above been described.Spendable enzyme activity toxin and fragment thereof comprise diphtheria toxin A chain, the non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa Pseudomonas aeruginosa), ricin (ricin) A chain, toxalbumin (abrin) A chain, capsule lotus root toxalbumin (modeccin) A chain, α-broom aspergillin (sarcin), tung oil tree (Aleutites fordii) toxalbumin, Dianthus caryophyllus L. (dianthin) toxalbumin, dyers' grapes (Phytolacaamericana) toxalbumin (PAPI, PAPII and PAP-S), balsam pear (Momordica charantia) inhibition, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) inhibition, white tree toxalbumin (gelonin), NSC-69529 (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (trichothecenes).Multiple radionuclide can be used for generating radiation coupling antibody.Example comprises ²¹²bi, ¹³¹i, ¹³¹in, ⁹⁰y and ¹⁸⁶re.

Can prepare with multiple bifunctional protein coupling agent by the conjugate of antibody and cytotoxic agent, such as N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP), imino-sulfane (IT), imido-ester (all example hydrochloric acid hexanedioyl imido acid dimethyl esters), active ester class (such as suberic acid two succinimido esters), aldehydes (such as glutaraldehyde), double azido compound (such as two (p-azido benzoyl base) hexanediamine), dual azepine derivatives (such as two (p-diazobenzene formyl radical) quadrol), vulcabond is (such as toluene 2, the 6-vulcabond), with the double activated fluorine cpd (such as 1, 5-bis-fluoro-2, the 4-dinitrobenzene) dual-function derivative.For example, can be as Vitetta et al., Science 238:1098 prepares the ricin immunotoxin described in (1987).The 1-isothiocyanic acid phenmethyl of carbon-14 mark-3-methyl diethylene triaminepentaacetic acid(DTPA) (MX-DTPA) is for the exemplary sequestrant by radioactive nuleus thuja acid and antibody coupling.Referring to WO 94/11026.

In another embodiment, can be by antibody and " acceptor " (such as streptavidin) thus coupling for tumour target in advance, wherein to patient's administration of antibodies-acceptor conjugate, then use scavenging agent to remove unconjugated conjugate in circulation, then use " part " (as the affinity element) with cytotoxic agent (as the radioactive nuleus thuja acid) coupling.

immunoliposome

Antibody disclosed herein also can be mixed with immunoliposome.Can prepare by means known in the art by the liposome that contains antibody, such as Epstein et al., and Proc.Natl.Acad.Sci.USA 82:3688 (1985); Hwang et al., Proc.Natl.Acad.Sci.USA 77:4030 (1980); U.S. Patent No. 4,485,045 and 4,544,545.The liposome that extend cycling time is disclosed in U.S. Patent No. 5,013,556.

Available packages generates useful especially liposome containing the lipid composite of phosphatidyl choline, cholesterol and PEG derivatization phospholipid acyl thanomin (PEG-PE) by reverse phase evaporation.Liposome is pushed through and has the filter of setting aperture, produce the liposome with desired diameter.Can, as Martin et al., described in J.Biol.Chem.257:286-288 (1982), the Fab ' fragment of antibody of the present invention be reacted and the liposome coupling through disulfide exchange.Optionally in liposome, comprise chemotherapeutics (such as Dx (Doxorubicin)).Referring to Gabizon et al., J.National Cancer Inst.81 (19): 1484 (1989).

the medicinal compositions of antibody and polypeptide

The antibody of the specific binding polypeptide that this paper identifies and other molecule of identifying by above-disclosed screening assay method, can use to treat above and various illnesss hereinafter described with the form of medicinal compositions.

Fat transfection agents (lipofectin) or liposome also can be used for polypeptide of the present invention and antibody or composition are delivered in cell.When using antibody fragment, the minimum inhibition fragment of the binding domains of preferred specific binding target protein.For example, the variable region sequences based on antibody, can design the peptide molecule retained in conjunction with the ability of target protein sequence.This type of peptide can chemosynthesis and/or is generated (referring to for example Marasco et al., Proc.Natl.Acad.Sci.USA 90:7889-7893 (1993)) by recombinant DNA technology.

Preparaton herein also can containing treating to some extent, concrete indication is necessary surpasses a kind of active compound, preferably active complementary and there is no each other a disadvantageous effect.Perhaps/in addition, composition also can comprise the medicament that strengthens its function, such as for example cytotoxic agent, chemotherapeutics or growth inhibitor.Suitable, this quasi-molecule exists effectively to measure combination for predetermined purpose.

Activeconstituents also can wrap and for example for example be stated from, for example, by (being respectively Walocel MT 20.000PV or gelatin microcapsule and poly-(methyl methacrylate) microcapsule) in condensation technique or the microcapsule that prepare by interfacial polymerization, in gluey drug delivery system (liposome, white protein microsphere, microemulsion, nano particle and Nano capsule) or in macro emulsion.This type of technology is disclosed in for example Remington ' sPharmaceutical Sciences, sees above.

The preparaton that is used for using in body must be aseptic.This can be easy to by realizing with aseptic membrane filtration.

Can prepare extended release preparation.The suitable example of extended release preparation comprises the solid hydrophobic polymkeric substance semipermeability matrix that contains antibody, and this matrix is the form of standardized product, for example film or microcapsule.The example of sustained release matrix comprises polyester, hydrogel (for example poly-(2-hydroxyethyl-methacrylic ester) or poly-(vinyl alcohol)), polylactide (United States Patent (USP) 3,773,919), the multipolymer of Pidolidone and Pidolidone γ-ethyl ester, nondegradable ethane-acetic acid ethyenyl, degradable lactic acid-ethanol copolymer are such as LUPRONDEPOT ^tM(the Injectable microspheres body formed by lactic acid-ethanol copolymer and leuprorelin acetate) and poly--D-(-)-3-hydroxybutyrate.Although polymkeric substance such as ethane-acetic acid ethyenyl and lactic acid-ethanol can discharge molecule and reach more than 100 days, the time of some hydrogel release protein is shorter.When encapsulated antibody maintains in vivo for a long time, they may owing to being exposed to the wet environment of 37 ℃, sex change or gathering cause biologic activity loss and immunogenicity to change.Can carry out stabilization strategy reasonable in design according to related mechanism.For example, if being the intermolecular S-S key exchanged via mercaptan-disulphide, discovery aggregation mechanism forms, so can be by modifying sulfhydryl residue, being realized stablizing by acidic solution freeze-drying, controlling moisture, the suitable additive of employing and exploitation particular polymers substrate composition.

diagnostic uses and imaging

Can be used for diagnostic purpose through the antibody of the specific binding polypeptide of mark and derivative thereof and analogue, detecting, diagnosis or monitoring disease and/or the illness relevant with expression, unconventionality expression and/or the activity of polypeptide of the present invention.According to a preferred embodiment, antibody of the present invention can be used for diagnostic assay method or imaging assay method, and it involves antibody is injected into to the experimenter.The invention provides the detection of VEGF or α 5 β 1 polypeptide unconventionality expressions, comprise that (a) used the expression of for example, in the cell (tissue) of one or more TPPA individualities of the present invention or body fluid polypeptide, and (b) gene expression dose and standard gene expression level are compared, thus the rising compared with the standard expression level of the gene expression dose of measuring or reduce the indication unconventionality expression.

Antibody of the present invention is also for measuring the protein level of biological sample, wherein use the classical immunohistology method that those skilled in the art will know that (referring to for example Jalkanen, et al., J.Cell.Biol.101:976-985 (1985); Jalkanen, et al., J.Cell.Biol.105:3087-3096 (1987)).Can be used for detecting other method based on antibody that protein gene expresses and comprise immunoassay, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).Suitable assay for antibodies marker is that this area is known, comprises the enzyme labelling thing, such as glucose oxidase; Radio isotope, such as iodine ( ¹³¹i, ¹²⁵i, ¹²³i, ¹²¹i), carbon ( ¹⁴c), sulphur ( ³⁵s), tritium ( ³h), indium ( ^115min, ^113min, ¹¹²in, ¹¹¹in), technetium ( ⁹⁹tc, ^99mtc), thallium ( ²⁰¹ti), gallium ( ⁶⁸ga, ⁶⁷ga), palladium ( ¹⁰³pd), molybdenum ( ⁹⁹mo), xenon ( ¹³³xe), fluorine ( ¹⁸f), ¹⁵³sm, ¹⁷⁷lu, ¹⁵⁹gd, ¹⁴⁹pm, ¹⁴⁰la, ¹⁷⁵yb, ¹⁶⁶ho, ⁹⁰y, ⁴⁷sc, ¹⁸⁶re, ¹⁸⁸re, ¹⁴²pr, ¹⁰⁵rh, ⁹⁷ru; Luminol,3-aminophthalic acid cyclic hydrazide; Fluorescent marker, such as fluorescein and rhodamine; And vitamin H.

Technology known in the art can be applicable to mark antibody of the present invention.This type of technology includes but not limited to use bifunctional coupling agent (referring to for example U.S. Patent No. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; 5,808,003, the contents intact of each piece is collected herein by reference).

Can comprise α 5 β 1 that detect in Mammals and/or the step of VEGF molecule with the diagnosis of VEGF and/or α 5 disease that β 1 expresses or unconventionality expression is relevant or illness in animal (preferred mammal, optimum is chosen).In one embodiment, after using the VEGF antagonist, diagnosis comprises: (a) to administration (for example parenteral, subcutaneous or intraperitoneal) significant quantity through mark anti-alpha 5 beta 1 antibody, (b) after using, wait for a period of time, allow through tagged molecule preferentially in the experimenter position of α 5 β 1 developed by molecule concentrate (and unconjugated remove to background level through tagged molecule); (c) measure background level; And (d) detect in the experimenter through tagged molecule, wherein detect through tagged molecule and suffer from β 1 expresses or unconventionality expression is relevant specified disease or illness with α 5 higher than background level indication experimenter.Background level can be measured by several different methods, comprises that the amount through tagged molecule will detected and the typical curve of measuring for particular system in advance compare.

According to a specific embodiment, α 5 β 1 expression of polypeptides or cross to express be diagnosis after using the agent of VEGF antagonist for treating or the prognosis assay method in measure, be evaluated at α 5 β 1 levels (for example, through the immunohistochemical methods assay method, using anti-alpha 5 beta 1 antibody) that exist on cell surface.Perhaps/in addition, can measure the nucleic acid of coding for alpha 5 β 1 polypeptide in cell or the level of mRNA, for example, through fluorescence in situ hybridization, use the probe based on nucleic acid, its nucleic acid corresponding to coding for alpha 5 β 1 or its complementary sequence (FISH; Referring to WO98/45479, be published in October, 1998), Southern trace, Northern trace or polymerase chain reaction (PCR) technology, such as real-time quantitative PCR (RT-PCR).Can also study α 5 β 1 by measurement biological fluid such as the released antigen in serum and cross expression, for example use the assay method based on antibody (also can, referring to for example U.S. Patent No. 4,933,294, to be announced June 12 nineteen ninety; WO91/05264, be published on April 18th, 1991; U.S. Patent No. 5,401,638, announced March 28 nineteen ninety-five; And Sias et al., J.Immunol.Methods 132:73-80 (1990)).Beyond the said determination method, multiple in vivoassay method can be for skilled practitioner.For example, cell in body of mammals can be exposed to antibody, it optionally uses the detectable label substance markers, radio isotope for example, and can assess the combination of antibody to cell in Mammals, for example by the external scan radioactivity or by analysis, take from the mammiferous biopsy that before is exposed to antibody.

By complete being collected herein by reference of all publications (comprising patent and patent application) quoted, specifically comprise the U.S. Provisional Application No.60/784 that on March 21st, 2006 submits to, 704 herein; The U.S. Provisional Application No.60/785 that on March 22nd, 2006 submits to, 330; With the U.S. Provisional Application No.60/871 submitted on December 22nd, 2006,743.

Following DNA sequence dna is preserved in American type culture collection (American Type Culture Collection (ATCC) according to the clause of budapest treaty, 10801 University Blvd., Manassas, VA 20110-2209, USA), as described below:

Material preserving number preservation day

α 5/ β 1 7H5.4.2.8 PTA-7421 on March 7th, 2006

α 5/ β 1 7H12.5.1.4 PTA-7420 on March 7th, 2006

This paper preservation is to carry out for the microbial preservation budapest treaty (Budapest Treaty) of patented procedure and the regulation of (budapest treaty) detailed rules for the implementation thereof according to international recognition.This has guaranteed from preservation to preserve the survival culture 30 years of preservation.Preserved material can obtain by ATCC according to the clause of budapest treaty, and the agreement between obedience Genentech company and ATCC, it guaranteed after relevant United States Patent (USP) mandate or at any U.S. or foreign patent application after public, with in both prior to the person be as the criterion, the public can be permanent and the offspring of unrestricted acquisition preservation culture, and guaranteed that the management article that reach according to it according to 35 U.S.C.122 (comprise 37 C.F.R.1.14, to mention 886OG 638 especially) by the individual of United States Patent and Trademark Office head approval by the offspring of qualified acquisition preservation culture.

The application's transferee agrees, if dead when the culture of preserved material is cultivated under conditions suitable, lose or destroyed, he will be after having notice rapidly with another part of material replacing of same culture.The availability of institute's preserved material also is not interpreted as and implements license of the present invention to violating the right that any government organs authorize according to its patent law.

Except as otherwise noted, the commercialization reagent of mentioning in embodiment is used according to the specification sheets of manufacturers.The source of those cells of hereinafter being differentiated with the ATCC numbering in embodiment and entire description is American type culture collection (American Type Culture Collection, Manassas, VA).Except as otherwise noted, the present invention uses the normal process of recombinant DNA technology, and such as above reaching those that put down in writing in following textbook: Sambrook et al. sees above; Ausubel et al., Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989; Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press, Inc., N.Y., 1990; Harlow et al., Antibodies:A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, 1988; Gait, Oligonucleotide Synthesis, IRL Press, Oxford, 1984; Freshney, Animal CellCulture, 1987; Coligan et al., Current Protocols in Immunology, 1991.

Run through entire description and claims, word " comprises " or its version, be understood to mean and comprise described integer/composition or integer group/one-tenth grouping such as " comprising " or " containing ", but do not get rid of any other integer/composition or integer group/one-tenth grouping.

Think that aforementioned written description is enough to make those skilled in the art can implement the present invention.Provide following embodiment only for the illustration purpose, be not intended to limit the scope of the invention by any way.In fact, according to top description, at this paper shown and describe outside, multiple modification of the present invention is apparent for those skilled in the art, and falls within the scope of the appended claims.

Embodiment

Embodiment 1: after anti-VEGF therapy, the stroma cell of express alpha 5 β 1 raises

Express dyeing by having used the section of the HT-29 Human colorectal carcinoma heterograft of VEGF antibody B20-4.1 monotherapy in athymic mouse for anti-alpha 5 beta 1.With the control group with control antibodies (anti-artemisiifolia antibody) treatment in this research, compare, the B20-4.1 monotherapy produces and very little or intermediate value that non-activity is corresponding time (time to endpoint, TTE) from home.Described tumour is carried out biweekly, continue the measurement of 58 days.When the tumour of animal reaches terminal volume 1000mm ³the time or in the time of 58 days, with the first comer, be as the criterion, they are implemented to euthanasia, and be every mouse calculating TTE.From per-cent tumor growth delay (%TGD), determine treatment result, %TGD is defined as: the intermediate value TTE for the treatment of mouse increases than the per-cent of the intermediate value TTE of contrast mouse, use Logrank to analyze, 0.01≤P≤0.05 is considered as significant difference, and P<0.01 is considered as highly significant difference.The intermediate value TTE value of control group is 20.6 days.The treatment of B20-4.1 monotherapy produces and there is no active 20.1 days corresponding intermediate value TTE.

Fig. 1 has shown the tumor biopsy with the anti-alpha 5 beta 1 antibody staining.Observing the stroma cell of increase after anti-VEGF treatment raises.These stroma cells are to beta 2 integrin alpha 5 β 1 positive (BG dyeing).

Embodiment 2: anti-alpha 5 beta 1 antibody

Give people α 5 β 1 (Chemicon CC1027) of injected in mice purifying.To express the plasmoma cellular segregation of anti-alpha 5 beta 1 antibody and be transformed into hybridoma cell line.Two hybridoma cell lines of called after 7H5.4.2.8 and 7H12.5.1.4 are preserved in to ATCC.See above.The antibody produced by the 7H5.4.2.8 hybridoma is mIgG2a κ antibody (here also referred to as " 7H5 antibody ").The antibody produced by the 7H12.5.1.4 hybridoma is mIgG2b κ antibody (here also referred to as " 7H12 antibody ").

The direct binding assay of embodiment 3:HUVEC

The tissue culture that will contain the Human umbilical vein endothelial cells (HUVEC) in growth with PBS cleans twice.With 3-4ml 5mM EDTA/PBS solution, cell is broken away from from culturing bottle.Fresh substratum is added to cell and mixes.Aliquots containig counting to the cell in this mixture.By cell centrifugation and with cleaning buffer solution (50mM Tris, 150mM NaCl, pH7.5), clean once.Adjust cell concn, make and cell can be inoculated into to 96 hole MSD height in conjunction with on flat board with the 25ul/ hole with 25,000 cells/well, perhaps 4,000 cells/well (is respectively Cat#L11XB-1 or #L11XB-2, Meso ScaleDiagnostics, LLC) on 384 hole flat boards.Cell is caught allowing in room temperature incubation 1 hour on flat board.For closed pores, 25ul is stored to damping fluid (at TBS (50mM Tris, 150mM NaCl)+1mM CaCl ₂/ 1mMMgCl ₂, 30% foetal calf serum (FBS) in pH7.5)) and add hand-hole and in room temperature incubation 30 minutes to 1 hour.

With measuring damping fluid (with 1mM CaCl ₂/ 1mMMgCl ₂tBS, pH7.2+2-4%FBS) by the serial dilution of anti-alpha 5 beta 1 antibody to there is Multiple Antibodies concentration.With cleaning buffer solution wash-out hole twice, then blot.The 25ul antibody diluent is added to hand-hole, then incubation on ice 1 hour.With TBS, hole is cleaned three times.

25ul 0.5ug/ml xmuFc-sulfo-tag solution is added to each hole and incubation on ice 45 minutes to 1 hour.XmuFc-sulfo-tag is mountain sheep anti-mouse igg (catalog number R23-AC-5).Add MSD-SA-tag (catalog number R32-21-AD-5), and incubation on ice 45 minutes to 1 hour.With TBS, hole is cleaned three times.150ul 2X reading damping fluid is added to each hole, and (4X MSD reads damping fluid, uses dH ₂o is diluted to 2X, cat # R92TD-1 (surfactant-free)).Electrochemiluminescence (ECL) signal is subsequently measured by photorectifier and used MSD reading apparatus (giving tacit consent to 6000 schemes) to be quantified as relative light unit.Fig. 2 has shown the result of the direct binding assay of HUVEC.The EC50 of 7H5 antibody is 0.22nM.The EC50 of 7H12 antibody is 0.38nM.

Embodiment 4: anti-alpha 5 beta 1 antibody FACS assay method

By 7H12 or 7H5 antibody and RAJI cell (the not clone of express alpha 5 β 1 mRNA) or HUVEC cell (expressing the clone of high-level α 5 β 1 mRNA) incubation in 100ul.Detect the cell of combination by the second antibody of fluorescence coupling.Fig. 3 shown by facs analysis, 7H12 and 7H5 in conjunction with the HUVEC cell not in conjunction with the RAJI cell.Constructed by using rabbit synovial cell (HIG-82) or rhesus monkey (rhesus monkey) cell (CL-160 macaque (macaca mulatta) inoblast or CRL-1780 retina endotheliocyte) to implement, we observe 7H12 and 7H5 in conjunction with rabbit and MC.Embodiment 5: while having anti-alpha 5 beta 1 antibody to the cell adhesion of fibronectin

Fibronectin (Sigma F1141 (ox) or Roche 1080938 (people)) is diluted to 1ug/ml in sodium carbonate buffer.100 μ l fibronectin solution are added to each hole of NUNC maxisorp 96 hole flat boards and allow it in 4 ℃ of combinations spend the night (the flat immunity in NUNC 96 holes is dull and stereotyped, MaxiSorp N/Ster439454 (VWR 62409-002)).Then phosphate buffered saline (PBS) for hole (PBS) is cleaned and uses 1%BSA (Sigma A9418) sealing at least 30 minutes.Then with PBS, this flat board is cleaned three times.By 20,000 HUVEC cells add each hole and with contain 1.4mM MgCl ₂with 1.4mM CaCl ₂growth medium in 7H5 or the 7H12 incubation of various concentration.Then this incubation mixture is added to the coated flat board of fibronectin.In the situation that does not add inhibiting antibody, about 20,000 cells in the isometric growth substratum are added to each control wells.

Flat board is rotated 5 minutes so that cell is synchronizeed with the contact of substrate with 140g.By cell at CO ₂cultivate various time spans (0 to 120 minute) in incubator.The length of incubation time is difference with various clone.Then with PBS, flat board is cleaned three times.All liquid is removed from hole and freezing in-80 ℃.Then in the room temperature flat board that thaws.CyQuant damping fluid (Molecular Probes CyQuant C7026) is added in hole and by flat board in room temperature incubation 10 minutes.Measure the OD reading.Fig. 4 has shown that the IC50 of 7H5 antibody is 0.85ug/ml (3.44nM), and the IC50 of 7H12 antibody is 0.7ug/ml (4.38nM).

Embodiment 6: the proliferation assay of using the HUVEC cell

96 holes flat boards are spent the night with fibronectin (1 μ g/ml) is coated.Then with PBS, clean dull and stereotyped.3000-5000 endotheliocyte (EC) added to each hole of 96 orifice plates and allow to be attached to fully hole.To resist α 5 antibody to add (comprising the isotype contrast).For every kind of condition is used 3 holes.Then by cell and antibody incubation 1-24 hour.For example, at some concentration (0 μ g/ml, 4 μ g/ml, 16 μ g/ml, 60 μ g/ml, 120 μ g/ml) test anti-alpha 2 integrin alpha 5 beta 1 antibodies.

Then cell is used to the BrdU mark, by by them and 2 μ l BrdU storing solutions (25mg/ml in PBS) incubation in 1ml tissue culture medium (TCM) (EGM2+ is from all fill-ins (Cat#CC-4176) of Clonetics).After this incubation, cell is fixed with 4%PFA, processed 20 minutes with 1N HCl, with PBS, clean several times, then sealing 1-2 hour in 10% lowlenthal serum (with the PBS of 0.2%Triton).Then cell used to monoclonal antibody (BD Cat#347580,1:40 dilution in the PBS with 0.2%Triton and the 5% lowlenthal serum) dyeing for BrdU and be incubated overnight in 4 ℃.Next day, cell is cleaned 3 times with PBS and in the dark in anti-rabbit (1:800) the second antibody incubation of room temperature and Alexa-594 coupling 4 hours.Hole is cleaned again and with DAPI (1:10 in PBS, 000 dilution) incubation 10 minutes.After cleaning with PBS is last, by the photo of taking DAPI dyeing at 5X, come the total cell count counting in every hole.With red filter in same field of view to BrdU positive cell take pictures.Propagation is evaluated as in this visual field the per-cent of positive cell to BrdU.Then carry out analytical results with Excel.Fig. 5 a has shown after 5000 initiator cell number in the HUVEC total cell count of 32 hours.Fig. 5 b has shown that in antibody concentration be in the HUVEC total cell count of 24 hours in 20ug/ml.

Embodiment 7: migration mensuration scheme

HUVEC is cultivated on coated 24 hole flat boards at 5 μ g/ml fibronectins in from all fill-ins (Cat#CC-4176) of Clonetics at EGM2+, until cell converges.Then by 2 μ l imbibition device tips, the cell of each hole central authorities is rule (scared) and washed away the cell of removing by line.To add with the cell culture medium of control antibodies, 7H5 or 7H12 different holes.Use the antibody of all tests at 20 μ g/ml.Then make cell allow growth 1 to 2 day.The monitoring wound area.Fig. 6 has shown at 0 hour and 30 hours, the HUVEC migration photo in the situation of anti-α 5 antibody of the 20ug/ml with in ECM-2 (7H5) on the 5ug/ml fibronectin.Fig. 7 is the figure the % migration of 30 hours with the cell of 7H5 or 7H12 antibody treatment.

Caspase-3 immunostaining apoptosis the assay method that embodiment 8:HUVEC activates

96 holes flat boards are spent the night with fibronectin (1 μ g/ml) is coated.With PBS, clean dull and stereotyped.Then, 3000-5000 HUVEC cell spread into each hole of 96 orifice plates and in perfect medium (with the EBM-2 substratum (Cambrex CC-3156) of EGM-2 SingleQuots (Cambrex CC-4176)) overnight incubation.If for the apoptosis assay method, this substratum is 50/50 substratum with 10%FBS so by the 2H-11 mouse endothelial cells.

Next day, serum free medium and incubation 4-6 hour are changed into in one group of hole so that cell is hungry and make them in nonproliferative state.Other group cells are remained in perfect medium and represent the cell in active propagation.After 4-6 hour, add antibody (comprising the isotype contrast).Generally speaking, use 3 holes for every kind of condition.Then by cell and antibody incubation 1-48 hour.Usually by the anti-alpha 2 integrin alpha 5 beta 1 antibodies at following concentration determination: 0 μ g/ml, 4 μ g/ml, 16 μ g/ml, 60 μ g/ml and 120 μ g/ml.

After this incubation, cell is fixed with 4%PFA, sealing 1-2 hour in 10% lowlenthal serum (with the PBS of 0.2%Triton), then for example, with the monoclonal antibody of identifying specifically the activated form of caspase 3 (from the rabbit activity resistent caspase 3 antibody of BioVision, 1:50 dilutes in the PBS with 0.2% Triton and 5% lowlenthal serum) dyeing.Anti-caspase 3 antibody and fixing cell are incubated overnight in 4 ℃.Next day, cell is cleaned 3 times with PBS and with the anti-rabbit second antibody (1:800) of Alexa-594 coupling in the dark in room temperature incubation 4 hours.Cell is cleaned again and with DAPI (1:10 in PBS, 000 dilution) incubation 10 minutes.After cleaning with PBS is last, by the photo of taking DAPI dyeing at 5X, come the total cell count counting in every hole.With red filter in same field of view to the activation the positive cell of caspase 3 take pictures.Apoptosis is evaluated as the per-cent of the cell positive to the caspase 3 of activation.Then carry out analytical results with Excel.It is apoptosis-induced that Fig. 8 has shown that 7H5 and 7H12 do not enliven.

The active colorimetric method of embodiment 9:HUVEC Caspase-3/7

Use 7H5 and 7H12 antibody (from Apo-One Caspase-3/7 assay method of Promega, referring to the technical bulletin No.295 of standard 96 hole assay method specification sheetss) to carry out caspase 3/7 activation measurements.

Generally speaking, with fibronectin (1 μ g/ml), dull and stereotyped being coated with in 96 holes spent the night.With PBS, clean dull and stereotyped.Then 3000-5000 HUVEC cell spread into each hole of 96 orifice plates and in perfect medium (with the EBM-2 substratum (CambrexCC-3156) of EGM-2 SingleQuots (Cambrex CC-4176)) overnight incubation.If for the apoptosis assay method, this substratum is 50/50 substratum with 10%FBS so by the 2H-11 mouse endothelial cells.

Next day, serum free medium and incubation 4-6 hour are changed into in one group of hole so that cell is hungry and make them in nonproliferative state.Other group cells are remained in perfect medium and represent the cell in active propagation.After 4-6 hour, add antibody (comprising the isotype contrast).Generally speaking, use 3 holes for every kind of condition.Then by cell and antibody incubation 24-48 hour.Usually by the anti-alpha 2 integrin alpha 5 beta 1 antibodies at following concentration determination: 0 μ g/ml, 4 μ g/ml, 16 μ g/ml, 60 μ g/ml and 120 μ g/ml.

After this incubation, 100 μ l Apo-One caspase 3s/7 reagent are added to every hole, then use dull and stereotyped mixing tank to mix 30 seconds so that 300rpm is gentle.Then then flat board is used and read the plate instrument in the room temperature incubation in 1 to 8 hour.Measure the fluorescence in every hole at 485nm excitation wavelength and 530nm emission wavelength.

Fluorescent signal (RLU) the indication apoptosis produced by the cutting of caspase 3/7 substrates.It is apoptosis-induced that Fig. 9 has shown that 7H5 and 7H12 do not enliven.

Embodiment 10: pipe forms assay method

Can resist the ability that their killer tubes of alpha 5 beta 1 antibodies assessment form.Following content is the example with the pipe formation assay method of managing the formation assay method based on the HUVEC rudiment of describing in Nakatsu et al. (2003) Microvascular Research 66 (2003) 102-112.

Generally speaking, HUVEC cell and the coated Cytodex3 microcarrier (Amersham Pharmacia Biogech, Piscataway, NJ) of dextran can be mixed in 1ml EGF-2 substratum with the concentration of 400 every pearls of HUVEC.At 37 ℃ and 5%CO ₂, continuing 4 hours, can within every 20 minutes, will gently vibrate with the pearl of cell.After incubation, the pearl with cell can be transferred to 25cm ²tissue culture flasks (BD Biosciences, Bedford, MA) and at 37 ℃ and 5%CO ₂, stop 12-16 hour in 5ml EGM-2.Next day, the pearl with cell can be cleaned three times and is resuspended in the concentration of the coated pearl/ml of 200 granulocytes with 1ml EGM-2 in 2.5 mg/ml Fibrinogens (Sigma, St.Louis, MO).500ul Fibrinogen/pearl solution can be added in to 0.625 unit zymoplasm (Sigma) in a hole of 24 hole tissue culture plate.Fibrinogen/pearl solution can condense 5 minutes in room temperature, then at 37 ℃ and 5%CO ₂condense 20 minutes.1ml EGM-2 (containing 2%FBS) can be added to each hole and at 37 ℃ and 5%CO ₂with fibrin clot balance 30 minutes.Substratum is removed from hole and changed with the 1ml fresh culture.About 20,000 skin flbroblast (Detroit 551, ATCC, Rockville, MD) can be layered on this grumeleuse above.Can example of spatial compartmentalizationis.Can monitor the pearl assay method 7 days.

Pearl that can HUVEC is coated is incubation 2-3 days in fibrin gel, on this gel with or without 500ul anti-alpha 5 beta 1 antibody (7H5 and 7H12), then transfer to the Stage microscope of the Nicon Eclipse TE300 that is equipped with multidimensional axle (multidimensional axes), and at 37 ℃ and 5%CO ₂maintain 72 hours.Can calculate by consideration fibrin gel volume the final antibody concentration that will use, i.e. final antibody concentration=total antibody quality/(culture volume+fibrin gel volume).Use Metamorph software within every 20 minutes, from a plurality of pearls, to catch image.Use the high-definition picture IX70 Olympus microscope of 4x object lens (for example with) of pearl to complete extracorporeal blood vessel quantitative.Compare with contrasting (untreated), can determine every bulbil number, wherein bud can be defined as to the blood vessel that length equals bead diameter.Can measure bud length by random unit.

Embodiment 11: the joint study in heterograft/allograft tumor model

Use and use in turn when can in heterograft/allograft tumor model, assess alpha 5 beta 1 antagonists therapy and VEGF antagonist therapy.Preferably, described model seldom or not responds VEGF antagonist therapy.Following content is the example of operable model: (a) the Fo5 allograft in the nude mouse (derived from the breast tumor of mmtv-Her2 transgenic mice) (Finkle, D., et al., (2004) Clin.Cancer Res.10:2499-2511); (b) the HT29 heterograft in nude mouse (people ties rectum system); (c) RIP-TbAg (pancreatic neoplasm in the Tg model).Be typically, can intraperitoneal, subcutaneous or intravenously uses described therapy.For example, VEGF antibody can be used 10mg/kg or administered twice 5mg/kg weekly once in a week.Based on its avidity and activity, can estimate the amount of alpha 5 beta 1 antagonists such as the antibody that will use.In an experiment, VEGF antagonist and alpha 5 beta 1 antagonists can be used to 5-6 week according to synchronized time schedule.Perhaps/in addition, can use in turn VEGF antagonist and alpha 5 beta 1 antagonists (for example, VEGF antibody is used three weeks, and then anti-alpha 5 beta 1 antibody dosed administration is three weeks).

Can assess therapeutic efficiency based on tumour progression, tumor perfusion (tumor perfusion), microvessel density, morphology and/or survival etc.Can measure tumour progression by for example gross tumor volume and/or tumor quality.The perfusion of FITC-lectin and vessel landmarks thing can be dyeed for assessment of the Vascular change of following tumour progression.

Embodiment 12:MDA-MB231 HBT model

To the female nude mice of HRLN at flank subcutaneous injection 5x10 ⁶individual MDA-MB231 human breast cancer cell.(HRLN is the strain name).Allow tumor growth, until they reach 80-120mm ³mean size.Then the mouse that will carry tumour is divided into 4 groups, and when the mean tumour volume of every group be about 100mm ³the time begin treatment.

During this research, weekly gross tumor volume is measured twice.The Application standard caliper measure carries out the gross tumor volume measurement.Anti-mouse beta 2 integrin alpha 5 monoclonal antibodies of hamster that are called 10E7 are produced at Genentech.When tumour is 1.5g or has pass by 60 days, with the first comer, be as the criterion, reach the terminal of this experiment.In some cases, to the respondent, follow the tracks of more of a specified duration.When reaching terminal, animal is implemented to euthanasia.

The processing details is described below:

(1) control group: inject anti-artemisiifolia contrast monoclonal antibody (10mg/kg, ip (intraperitoneal, interperitoneally), weekly)

(2) the single medicament group of anti-VEGF: injection anti-VEGF mAb B20.4.1 (10mg/kg, ip, weekly)

(3) combine group: B20.4.1 (10mg/kg, ip, the anti-mouse beta 2 integrin alpha 5 monoclonal antibody 10E7 of weekly)+hamster (10mg/kg, ip, biweekly)

(4) the single medicament group of anti-alpha 2 integrin α 5: the anti-mouse beta 2 integrin alpha 5 monoclonal antibody 10E7 of injection hamster (10mg/kg, ip, biweekly)

The control group data:

The single medicament group of anti-VEGF data:

Anti-VEGF and anti-alpha 5 beta 1 data:

The single medicament group of anti-alpha 2 integrin α 5 data:

This preliminary data has shown that the anti-VEGF of anti-α 5+ combines active early indication.

After the terminal of this research, calculated the mean tumour volume (Figure 11 A) of every group.Also drawn the Kaplan-Meier graphic representation to show the residue animal per-cent in this research, as the function (Figure 11 B) of time.Data presentation, the anti-alpha 2 integrin alpha 5 beta 1 antibodies has strengthened the effect of anti-VEGF in breast cancer model.

Embodiment 13: 7H12 and rhuMAb-VEGF in family's rabbit ear wound healing model

New Zealand white rabbit is weighed and use isoflurane (isofluorane) anesthesia.In every rabbit, from internal surface and along two auricle edges by trimming cropping.Any remaining hair is removed from operative site by the depilation washing lotion.Povidone iodine for operative site (betadine scrub) is clean, and then alcohol rinses.Use Aseptic technique, circular 8mm punching biopsy device is used for producing a wound to the credulous bone degree of depth at each ear.Remove perichondrium below with periosteum elevator and meticulous scissors.Will

elastoplast is positioned on each wound, and allows this rabbit from anesthesia recovery.

Remove every day

dressing, check wound, and surface applications is processed, and applies fresh dressing.The 0th (after operation immediately), 7,10,14 and 18 days, calculate the wound breach by measuring wound diameter.

Treatment group is:

100ug rhuMAb-VEGF (VEGF antibody) in each wound 30ul, (n=4) once a day

100ug 7H12 in each wound 30ul (anti-alpha 5 beta 1 antibody), (n=4) once a day

100ug 7H2 in 100ug rhuMAb-VEGF+15ul in each wound 15ul, (n=4) once a day

100ug trastuzumab (Anti-HER 2) in each wound 30ul, (n=3) once a day

Data presentation anti-VEGF and anti-alpha 5 beta 1 conjoint therapy there is surprising effect (Figure 10) than independent single medicament in this blood vessel generation model.

Embodiment 14: the anti-alpha 5 beta 1 in colorectal carcinoma+anti-VEGF conjoint therapy

To the female nu/nu mouse of HRLN at flank subcutaneous injection 1mm ³hT29 tumor fragment (colon tumor).Allow tumor growth, until they reach 80-120mm ³mean size, use afterwards therapy for treating.Then the mouse that will carry tumour is divided into 4 groups:

After the terminal of this research, calculated the mean tumour volume (Figure 12 A) of every group.Also drawn the Kaplan-Meier graphic representation to show the residue animal per-cent in this research, as the function (Figure 12 B) of time.Data presentation, the anti-alpha 2 integrin alpha 5 beta 1 antibodies has strengthened the effect of anti-VEGF in model of colon cancer.

Embodiment 15: the anti-alpha 5 beta 1+chemotherapy in colorectal carcinoma

To the female nu/nu mouse of HRLN at flank subcutaneous injection 5x10 ⁶individual HCT116 tumour cell (colon tumor cell).Allow tumor growth, until they reach 80-120mm ³mean size, use afterwards therapy for treating.Then the mouse that will carry tumour is divided into 4 groups:

The Application standard caliper measure biweekly carries out the gross tumor volume measurement.Anti-mouse beta 2 integrin alpha 5 monoclonal antibodies of hamster that are called 10E7 produce at Genentech.Body weight was measured 5 times in 2 days, then weekly twice (biwk) until this research finish.The terminal of experiment is the gross tumor volume of 1.5g or 60 days, with the first comer, is as the criterion.To some respondents, follow the tracks of more of a specified duration.When reaching terminal, animal is implemented to euthanasia.The dosed administration volume is 10mL/kg (0.200ml/20g mouse), according to body weight, adjusts this volume.Within 30 minutes after using 10E7, use irinotecan (irinotecan).For the animal that shows disappear fully (CR), the terminal that is organized in of tumour implant site is collected and is kept in formalin, follow 70%EtOH, for research subsequently.To treat that freezing all samples is placed in freezing mould, wraps up it and quick-frozen on liquid nitrogen in paper tinsel.

After the terminal of this research, calculated the mean tumour volume (Figure 13 A) of every group.Also drawn the Kaplan-Meier graphic representation to show the residue animal per-cent in this research, as the function (Figure 13 B) of time.Data presentation, the anti-alpha 2 integrin alpha 5 beta 1 antibodies does not strengthen the activity of chemotherapeutics (irinotecan) in model of colon cancer, but it does not hinder the activity of chemotherapeutics (irinotecan) yet.This observations and we are consistent about the conviction that (generally speaking, particularly the anti-angiogenic generation in the oncology background in), blood vessel injury should occur before significantly useful in anti-angiogenic generation in the anti-alpha 5 beta 1 therapy.By the VEGF antagonist such as

antibody can cause this blood vessel injury.Individually, chemotherapeutics does not cause significant blood vessel injury in this model.Use when it is contemplated that all these medicaments (VEGF antagonist/alpha 5 beta 1 antagonists/chemotherapeutics) or in turn, make and exist the VEGF antagonist to cause blood vessel injury.

Embodiment 16: α 5 β 1 Scatchard graphic representations

By the Iodogen method by the iodate of anti-alpha 5 beta 1 antibody, and use the PD-10 post by gel-filtration by radiolabeled antibody from free ¹²⁵the I-Na purifying.By the R9ab cell, rabbit fibroblast (purchased from ATCC, No.CCL-193) in 24 hole flat boards with approximately 50,000 every holes inoculations, and in 37 ℃ at 5% CO ₂middle cultivation 48 hours.With binding buffer liquid, (the 50:50 DMEM/F12 substratum that contains 2% FBS and 50mM HEPES, pH7.2) clean cell three times, then incubation on ice 15 minutes.By the cell after cleaning on ice with about 50pM ¹²⁵i-anti-alpha 5 beta 1 monoclonal antibody incubation 4 hours, should ¹²⁵the unmarked anti-alpha 5 beta 1 monoclonal antibody that I-anti-alpha 5 beta 1 monoclonal antibody contains the concentration of successively decreasing, this unmarked anti-alpha 5 beta 1 monoclonal antibody is diluted to continuously the concentration of 13 three repeated tests in binding buffer liquid from 0.5uM.With binding buffer liquid, cell is cleaned three times, then use 200ul SDS lysis buffer (the 100mM glycine, pH 3.0 for 1%SDS, 8M urea) to dissolve.The molten born of the same parents' thing of cell is counted on Wallac Wizard1470 gamma counter.Use the program NewLigand assessment of Genentech in conjunction with data, NewLigand is used Munson and Robard (Munson, P.and Robard, D. (1980) Anal.Biochem.107:220-239) curve fitting algorithm is with the binding affinity of determining antibody and the concentration of binding site.Figure 14 and 15 has shown that, in these binding assays, 7H5 antibody has the Kd of 0.10nM, and 7H12 antibody has the Kd of 0.30nM.

Embodiment 17: anti-alpha 2 integrin α 5 β 1 IgG epitope mappings/competitive binding assay method

At first by three times of serial dilution things of anti-alpha 2 integrin α 5 β 1 IgG in PBST damping fluid (PBS and 0.5% (w/v) BSA and 0.05% (v/v) Tween20) with human beta 2 integrin alpha 5 beta 1-6 antigen (1ug/ml; R& D) 96 coated hole Nunc Maxisorp flat boards are in room temperature incubation 1-2 hour, then add the biotinylated h7H5.v1 hIgG1 of 0.3nM (by Genentech, Inc. the 7H5 antibody variants produced) incubation is 15 minutes, and 0.3nM biotinylation h7H5.v1 hIgG1 is definite by inferior maximum combined signal (50-70%).Then use PBT damping fluid (PBS and 0.05% (v/v) Tween20) to clean this flat board 5 times.The streptavidin horseradish peroxidase thing (Pierce) that combining biotinylation h7H5.v1 hIgG1 is used in 1:2500 dilution in the PBST damping fluid detects, with TMB (TMB, Kirkegaard & Perry Labs, Gaithersburg, MD) substrate develops the color about 5 minutes, uses 1.0MH ₃pO ₄cancellation, and with spectrophotometry at the 450nm reading.With four parametrical nonlinearity regression curve fit procedure (Kaleidagraph, Synergy Software) matched curve.

Figure 16 has shown that the h7H5.v1 of combination is subject to the competition of the cold m7H5 of incremental change.In fact, the m7H5 competition curve almost is equal to h7H5.v1 competition curve (data do not show).Cold m7H12 also with vitamin H-h7H5.v1 competition α 5 β 1 combinations, show that h7H5.v1 and m7H12 are overlapping in conjunction with epi-position on α 5 β 1.On the other hand, control antibodies is not competed with the h7H5.v1 of combination.

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Claims (54)

1. an antibody, it comprises heavy chain variable domain and light chain variable territory, this heavy chain variable domain and light chain variable territory comprise six HVRs corresponding with six hypervariable regions (HVR) of the anti-alpha 5 beta 1 antibody generated by following hybridoma, the combination of anti-alpha 5 beta 1 antibody on human α 5 β 1 that wherein said antibody capable is generated by following hybridoma in conjunction with people α 5 β 1 and competitive inhibition, wherein said hybridoma be selected from March 7th, 2006 at ATCC as the hybridoma of ATCC No.PTA-7421 preservation with as the hybridoma of ATCC No.PTA-7420 preservation.

2. an antibody, heavy chain variable domain (VH) and light chain variable territory (VL) sequence that it comprises the antibody generated as the hybridoma of ATCC No.PTA-7421 preservation at ATCC by March 7th, 2006.

3. an antibody, heavy chain variable domain (VH) and light chain variable territory (VL) sequence that it comprises the antibody generated as the hybridoma of ATCC No.PTA-7420 preservation at ATCC by March 7th, 2006.

4. according to the antibody of claim 1-3 any one, wherein this antibody is humanized antibody or chimeric antibody.

5. according to the antibody of claim 1, wherein this antibody capable with the Kd between 500nM and 1pM in conjunction with people α 5 β 1 or α 5.

6. according to the antibody of claim 1, wherein this antibody can not be in conjunction with α V β 3 or α V β 5 or α V β 1.

7. according to the antibody of claim 1-3 any one, the Fc sequence that wherein this antibody comprises human IgG.

8. according to the antibody of claim 7, wherein said human IgG is IgG1 or IgG4.

9. according to the antibody of claim 7, the Fc sequence that wherein this antibody comprises cytotoxicity (ADCC) effector functions that lacks antibody dependent cellular.

10. according to the antibody of claim 1-3 any one, wherein this antibody is selected from Fab, Fab ', F (ab) ' ₂, scFv (scFv), Fv fragment and double antibody.

11., according to the antibody of claim 1-3 any one, wherein this antibody is linear antibody.

12., according to the antibody of claim 1-3 any one, wherein this antibody is multi-specificity antibody.

13. a conjugate, it comprises antibody and therapeutical agent according to claim 1-12 any one.

14., according to the conjugate of claim 13, wherein said therapeutical agent is selected from cytotoxic agent, radio isotope and chemotherapeutics.

15. a conjugate, it comprises antibody and marker according to claim 1-12 any one.

16., according to the conjugate of claim 15, wherein said marker is selected from radio isotope, fluorescence dye and enzyme.

17. the nucleic acid molecule of a separation, heavy chain variable domain (VH) and/or light chain variable territory (VL) of any antibody of its coding claim 1-12.

18. an expression vector, the nucleic acid molecule that it comprises claim 17.

19. a cell, the nucleic acid molecule that it comprises claim 17.

20., according to the cell of claim 19, wherein said cell is on March 7th, 2006 at ATCC as the hybridoma of ATCC No.PTA-7421 preservation or as the hybridoma of ATCC No.PTA-7420 preservation.

21. a method of producing antibody, comprise that (a) cultivates the cell of the nucleic acid molecule of the antibody that comprises coding claim 1-12 any one, and (b) reclaim the antibody by this Hemapoiesis.

22. a composition, antibody and pharmaceutical acceptable carrier that it comprises claim 1-12 any one.

23., wherein detect and comprise and make antibody contact sample, and detect the anti-alpha 5 beta 1 antibody that is bonded to α 5 β 1 protein for the preparation of detecting from the purposes in the reagent of α 5 β 1 protein in patient's sample according to the antibody of claim 1-12 any one.

24., according to the purposes of claim 23, wherein said antibody is in immunohistochemical methods (IHC) assay method or use in the ELISA assay method.

25. the purposes of antibody in the medicine of the blood vessel generation for the preparation of in suppressing the experimenter and/or vascular permeability according to claim 1-12 any one.

26., according to the purposes of antibody in the medicine of the disease for the preparation of in the treatment experimenter of claim 1-12 any one, wherein said disease has abnormal vascular and occurs or vascular permeability.

27. the blood vessel according to the antibody of claim 1-12 any one in the experimenter for the preparation of suppressing to suffer from disease occurs and/or the medicine of vascular permeability in purposes, wherein said medicine and VEGF antagonist are used in combination, and described disease has abnormal vascular and occurs or vascular permeability.

28. the purposes of claim 27, wherein said disease is selected from cancer, illness in eye and autoimmune disease.

29., according to the purposes of claim 27, first use the VEGF antagonist, rear administration of antibodies wherein to described experimenter.

30., according to the purposes of claim 27, use VEGF antagonist and antibody wherein to described experimenter simultaneously.

31., according to the purposes of claim 27, wherein the experimenter is used to the VEGF antagonist for treating, until then the experimenter uses Antybody therapy by the experimenter to the not response of VEGF antagonist for treating.

32., according to the purposes of claim 27, wherein said disease is cancer and by experimenter's standby VEGF antagonist for treating when described cancer right and wrong are invasive, and at described cancer standby Antybody therapy while being invasive.

33., according to the purposes of claim 27, wherein said experimenter compares α 5 β 1 levels with rising in illing tissue with the tissue from not suffering from this sick experimenter.

34., according to the purposes of claim 27, further use the therapeutical agent that is selected from lower group wherein to the experimenter: antineoplastic agent, chemotherapeutics, growth inhibitor and cytotoxic agent.

35., according to the purposes of claim 27, wherein said VEGF antagonist is VEGF antibody, wherein said VEGF antibody is subject to the competitive inhibition of rhuMAb-VEGF to the combination of people VEGF.

36., according to the purposes of claim 27, wherein said VEGF antagonist is VEGF antibody.

37., according to the purposes of claim 27, wherein said antibody coupling is to cytotoxic agent.

38., according to the purposes of claim 37, wherein said cytotoxic agent is radio isotope, chemotherapeutics or toxin.

39., according to the purposes of claim 36, wherein said VEGF antibody is rhuMAb-VEGF.

40., according to the purposes of claim 36, wherein said VEGF antibody is humanized antibody or people's antibody.

41., according to the purposes of claim 27, wherein said antibody is humanized antibody or people's antibody.

42. a composition, its antibody that comprises claim 1-12 any one, VEGF antagonist, and pharmacopedics can accept carrier.

43. the test kit for detection of α 5 β 1 in the experimenter who has crossed with the VEGF antagonist for treating, the antibody that it comprises claim 1-12 any one, and use the specification sheets of α 5 β 1 in the experimenter that this antibody test crossed with the VEGF antagonist for treating.

44. according to the composition of claim 42 for the preparation of suppress to suffer from have that abnormal vascular occurs or the experimenter of the disease of vascular permeability in blood vessel occur and/or the medicine of vascular permeability in purposes.

45., according to the purposes of claim 44, wherein said disease is selected from cancer, illness in eye, autoimmune disease.

46. according to the purposes of claim 27 or 44, wherein said disease is selected from solid tumor, metastatic tumor, soft tissue neoplasm, have the disease of a neovascularization, have inflammatory diseases that abnormal vascular occurs, be implanted into the disease occurred after the experimenter and have the disease of fiber vascular tissue abnormality proliferation.

47. the purposes according to claim 28 or 45, wherein said disease is cancer, and wherein said cancer is selected from mammary cancer, cervical cancer, colorectal carcinoma, lung cancer, non_hodgkin lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, soft tissue cancer and multiple myeloma.

48. the purposes according to claim 27 or 44, wherein said disease is selected from retinopathy, the macular degeneration that age brings out, rubescent, psoriatic, the inflammatory ephrosis, hemolytic uremic syndrome, diabetic nephropathy, sacroiliitis, inflammatory bowel, chronic inflammatory diseases, chronic detachment of retina, the cornea neovascularization, Crohn disease, myopia, eye neovascularity disease, Paget's disease, pemphigoid, polyarteritis, siogren's syndrome, ulcerative colitis, transplant rejection, pneumonia, nephrotic syndrome, oedema, the ascites relevant with malignant tumour, apoplexy, hemangiofibroma and neovascular glaucoma.

49., according to the purposes of claim 27 or 44, wherein said disease is psoriatic arthritis, corneal graft neovascularization, chronic uveitis, chronic hyalitis, corneal graft rejection or osteoarthritis.

50. the blood vessel according to the composition of claim 22 or 42 in the experimenter for the preparation of suppressing to suffer retina neovascularization after laser radiation shape keratomy occurs and/or the medicine of vascular permeability in purposes.

51. the blood vessel according to the composition of claim 22 or 42 in the experimenter for the preparation of suppressing to suffer the retina neovascularization occurs and/or the medicine of vascular permeability in purposes.

52. the purposes of the antibody of claim 1-12 any one in the experimenter's who suffers from disease for the preparation for the treatment of medicine, wherein said experimenter, once to by VEGF antagonist for treating disease, response being arranged, still partly has response or no longer includes response the VEGF antagonist now.

53., according to the purposes of claim 52, wherein said experimenter compares α 5 β 1 levels with rising in illing tissue with the tissue from not suffering from this sick experimenter.

54., according to the purposes of claim 52, further use the therapeutical agent that is selected from lower group wherein to the experimenter: antineoplastic agent, chemotherapeutics, growth inhibitor and cytotoxic agent.

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